Switch-region: target and method for inhibition of bacterial RNA polymerase

ABSTRACT

The invention provides a target and methods for specific binding and inhibition of RNA polymerase from bacterial species. The invention provides methods for identifying agents that bind to a bacterial RNA polymerase, and that inhibit an activity of a bacterial RNA polymerase, through interactions with a bacterial RNA polymerase homologous switch-region amino-acid sequence. Said methods comprise preparing a reaction solution comprising the compound to be tested and an entity containing a bacterial RNAP homologous switch-region amino-acid sequence, and detecting binding or inhibition. The invention has applications in control of bacterial gene expression, control of bacterial viability, control of bacterial growth, antibacterial chemistry, and antibacterial therapy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional application: 60/651,227filed Feb. 10, 2005, the contents of which are incorporated herein byreference.

GOVERNMENT SUPPORT

This invention was supported with U.S. Government funds (NIHRO1-GM41376). Therefore, the Government may have rights in theinvention.

BACKGROUND ART

Bacterial infections remain among the most common and deadly causes ofhuman disease. Infectious diseases are the third leading cause of deathin the United States and the leading cause of death worldwide (Binder etal. (1999) Science 284, 1311-1313). Multi-drug-resistant bacteria nowcause infections that pose a grave and growing threat to public health.It has been shown that bacterial pathogens can acquire resistance tofirst-line and even second-line antibiotics (Stuart B. Levy, TheChallenge of Antibiotic Resistance, in Scientific American, 46-53(March, 1998); Walsh, C. (2000) Nature 406, 775-781; Schluger, N. (2000)Int. J. Tuberculosis Lung Disease 4, S71-S75; Raviglione et al., (2001)Ann. NY Acad. Sci. 953, 88-97). New approaches to drug development arenecessary to combat the ever-increasing number of antibiotic-resistantpathogens.

The present invention provides one such approach.

RNA polymerase (RNAP) is the molecular machine responsible fortranscription and is the target, directly or indirectly, of mostregulation of gene expression (Ebright, R. (2000) J. Mol. Biol. 304,687-698; Darst, S. (2001) Curr. Opin. Structl. Biol. 11, 155-162;Cramer, P. (2002) Curr. Opin. Structl. Biol. 12, 89-97; Murakami & Darst(2003) Curr. Opin. Structl. Biol. 13, 31-39; Borukhov & Nudler (2003)Curr. Opin. Microbiol. 6, 93-100; Landick, R. (2001) Cell 105, 567-570;Korzheva & Mustaev (2001) Curr. Opin. Microbiol. 4, 119-125; Armache, etal. (2005) Curr. Opin. Structl. Biol. 15, 197-203; Woychik & Hampsey(2002); Cell 108, 453-463; Asturias, F. (2004) Curr. Opin. Genet Dev.14, 121-129; Cramer, P. (2004) Curr. Opin. Genet. Dev. 14, 218-226;Geiduschek & Kassavetis (2001) J. Mol. Biol. 310, 1-26). Bacterial RNAPcore enzyme has a molecular mass of ˜380,000 Da and consists of one β′subunit, one β subunit, two α subunits, and one ω subunit; bacterialRNAP holoenzyme has a molecular mass of ˜450,000 Da and consists ofbacterial RNAP core enzyme in complex with the transcription initiationfactor σ (Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S.(2001) Curr. Opin. Structl. Biol. 11, 155-162; Cramer, P. (2002) Curr.Opin. Structl. Biol. 12, 89-97; Murakami & Darst (2003) Curr. Opin.Structl. Biol. 13, 31-39; Borukhov & Nudler (2003) Curr. Opin.Microbiol. 6, 93-100). Bacterial RNAP core subunit sequences areconserved across Gram-positive and Gram-negative bacterial species(Ebright, R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr.Opin. Structl. Biol. 11, 155-162; Iyer, et al. (2004) Gene 335, 73-88).Eukaryotic RNAP I, RNAP II, and RNAP III contain counterparts of allbacterial RNAP core subunits, but eukaryotic-subunit sequences andbacterial-subunit sequences exhibit only limited conservation (Ebright,R. (2000) J. Mol. Biol. 304, 687-698; Darst, S. (2001) Curr. Opin.Structl. Biol. 11, 155-162; Cramer, P. (2002) Curr. Opin. Structl. Biol.12, 89-97).

Bacterial RNAP is a proven target for antibacterial therapy (Chopra, etal. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) TrendsBiochem. Sci. 29, 159-162). The suitability of bacterial RNAP as atarget for antibacterial therapy follows from the fact that bacterialRNAP is an essential enzyme (permitting efficacy), the fact thatbacterial RNAP subunit sequences are conserved (providing a basis forbroad-spectrum activity), and the fact that bacterial RNAP subunitsequences are only weakly conserved in eukaryotic RNAP I, RNAP II, andRNAP III (providing a basis for therapeutic selectivity).

The rifamycin antibacterial agents—notably rifampicin, rifapentine, andrifabutin—function by binding to and inhibiting bacterial RNAP (Chopra,et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) TrendsBiochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632;Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005)Cell 122, 351-363). The rifamycins bind to a site on bacterial RNAPadjacent to the RNAP active center and sterically and/or allostericallyprevent extension of RNA chains beyond a length of 2-3 nt (Chopra, etal. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) TrendsBiochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632;Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005)Cell 122, 351-363). The rifamycins are in current clinical use intreatment of Gram-positive and Gram-negative bacterial infections(Chopra, et al. (2002) J. Appl. Microbiol. 92, 4S-155; Darst, S. (2004)Trends Biochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105,621-632; Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al.(2005) Cell 122, 351-363). The rifamycins are of particular importancein treatment of tuberculosis; the rifamycins are first-lineanti-tuberculosis agents and are the only anti-tuberculosis agents ablerapidly to clear infection and prevent relapse (Mitchison, D. (2000)Int. J. Tuberc. Lung Dis. 4, 796-806). The rifamycins also are ofimportance in treatment of bacterial infections relevant to biowarfareor bioterrorism; combination therapy with ciprofloxacin, clindamycin,and rifampicin was successful in treatment of inhalational anthraxfollowing the 2001 anthrax attacks (Mayer, et al. (2001) JAMA 286,2549-2553), and combination therapy with ciprofloxacin and rifampicin,or doxycycline with rifampicin, is recommended for treatment of futurecases of inhalational anthrax (Centers for Disease Control andPrevention (2001) JAMA 286, 2226-2232).

The clinical utility of the rifamycin antibacterial agents is threatenedby the existence of bacterial strains resistant to rifamycins (Chopra,et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S. (2004) TrendsBiochem. Sci. 29, 159-162; Floss & Yu (2005) Chem. Rev. 105, 621-632;Campbell, et al. (2001) Cell 104, 901-912; Artsimovitch, et al. (2005)Cell 122, 351-363). Resistance to rifamycins typically involvessubstitution of residues in or immediately adjacent to the rifamycinbinding site on bacterial RNAP—i.e., substitutions that directlydecrease binding or function of rifamycins. A significant and increasingpercentage of cases of tuberculosis are resistant to rifampicin (1.4% ofnew cases, 8.7% of previously treated cases, and 100% of casesdesignated multidrug-resistant, in 1999-2002; Schluger, N. (2000) Int.J. Tuberc. Lung Dis. 4, S71-S75; Raviglione, et al. (2001) Ann. N.Y.Acad. Sci. 953, 88-97; Zumia, et al. (2001) Lancet Infect. Dis. 1,199-202; Dye, et al. (2002) J. Infect. Dis. 185, 1197-1202; WHO/IUATLD(2003) Anti-tuberculosis drug resistance in the world: third globalreport (WHO, Geneva)). Strains of bacterial bioweapons agents resistantto rifampicin can be, and have been, constructed (Lebedeva, et al.(1991) Antibiot. Khimioter. 36, 19-22; Pomerantsev, et al. (1993)Antibiot. Khimioter. 38, 34-38; Volger, et al. (2002) Antimicrob. AgentsChemother. 46, 511-513; Marianelli, et al. (204) J. Clin. Microbiol. 42,5439-5443).

In view of the public-health threat posed by rifamycin-resistant andmultidrug-resistant bacterial infections, there is an urgent need fornew classes of antibacterial agents that (i) target bacterial RNAP (andthus have the same biochemical effects as rifamycins), but that (ii)target sites within bacterial RNAP distinct from the rifamycin bindingsite (and thus do not show cross-resistance with rifamycins). (SeeChopra, et al. (2002) J. Appl. Microbiol. 92, 4S-15S; Darst, S (2004)Trends Biochem. Sci. 29, 159-162.)

Recently, crystallographic structures have been determined for bacterialRNAP and eukaryotic RNAP II (Zhang et al., (1999) Cell 98, 811-824;Cramer et al., (2000) Science 288, 640-649; Naryshkin et al., (2000)Cell 101, 601-611; Kim et al., (2000) Science 288, 1418-1421; Korzhevaet al., (2000) Science 289, 619-625; Ebright, R. (2000) J. Mol. Biol.304, 687-689; Cramer et al., (2001) Science 292, 1863-1876; Gnatt etal.,(2001) Science 292, 1876-1882; Mekler et al., (2002) Cell 108,599-614; Murakami et al., (2002) Science 296, 1280-1284; Murakami etal., (2002) Science 296, 1285-1290; Vassylyev et al., (2002) Nature 417,712-719; Bushnell et al., (2004) Science 303, 983-988; Westover et al.,(2004) Science 303, 1014-1016; Armache, et al., (2003) Proc. Natl. Acad.Sci. USA 100, 6964-6968). Moreover, cryo-EM structures have beendetermined for bacterial RNAP and eukaryotic RNAP I (Opalka, et al.(2000) Proc. Natl. Acad. Sci. USA 97,617-622; Darst, et al. (2002) Proc.Natl. Acad. Sci. USA 99, 4296-4301; DeCarlo, et al. (2003) J. Mol. Biol.329, 891-902).

Structures also have been determined for RNAP complexes with nucleicacids, nucleotides and inhibitors (Campbell, et al. (2001) Cell 104,901-912; Artsimovitch, et al. (2005) Cell 122, 351-363; Campbell, et al.(2005) EMBOJ. 24, 674-682; Artsimovitch, et al. (2004) Cell 117,299-310; Tuske, et al. (2005) Cell 122, 541-522; Temiaov, et al. (2005)Mol. Cell 19, 655-666; Vassulyev, et al. (2005) Nature Structl. Biol.12, 1086-1093; Gnatt, et al. (2001) Science 292, 1876-1882; Westover, etal. (2004a) Science 303, 1014-1016; Westover, et al. (2004b) Cell 119,481-489; Ketenberger, et al. (2004) Mol. Cell 16, 955-965; Bushnell, etal. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 1218-1222; Kettenberger, etal. (2005) Natl. Structl. Mol. Biol. 13, 44-48).

The structures reveal that RNAP-bacterial or eukaryotic-has a shapereminiscent of a crab claw. The two “pincers” of the “claw” define theactive-center cleft that can accommodate a double-stranded nucleicacid-and which has the active-center Mg²⁺ at its base (FIG. 1A). Thelargest submit (β′ in bacterial RNAP) makes up one pincer, termed the“clamp,” and part of the base of the active-center cleft. Thesecond-largest subunit (β in bacterial RNAP) makes up the other pincerand part of the base of the active-center cleft.

The structures further reveal that the RNAP clamp can exist in a rangeof distinct conformational states-from a fully open clamp conformationthat permits unimpeded entry and exit of DNA to a fully closed clampconformation that prevents entry and exit of DNA (FIG. 1A; Ebright(2000) J. Mol. Biol. 304, 687-698; Darst (2001) Curr. Opin. Structl.Biol. 11, 155-162; Cramer (2002) Curr. Opin. Structl. Biol. 12, 89-97;Murakami & Darst (2003) Curr. Opin. Structl. Biol. 13, 31-39; Borukhob &Nudler (2003) Curr. Opin. Microbiol. 6, 93-100; and Landick (2001) Cell105, 567-570). It has been proposed that the clamp opens to permit DNAto enter the active-center cleft in transcription initiation, closesafter DNA enters the active-center cleft in transcription initiation,and further closes, or acquires further stability in the closed state,in transcription elongation. Clamp closure is proposed to be responsiblefor the high stability of initiation complexes and for the exceptionallyhigh stability and exceptionally high processivity of elongationcomplexes.

The “switch region” is located at the base of the clamp (FIG. 1A). Theswitch region serves as a hinge that permits rotation of the β′ “pincer”relative to the remainder of RNAP, and, correspondingly, permits openingor closing of the RNAPactive-center cleft. The switch region adoptsdifferent conformations when the β′ pincer is rotated out of theactive-center cleft (open-clamp state, required for entry of DNA intoactive-center cleft) and when the β′ is rotated into the active-centercleft (closed-clamp state, required for stable binding of DNA withinactive-center cleft) (FIG. 1B; Cramer (2002) Curr. Opin. Structl. Biol.12, 89-97; Landick (2001) Cell 105, 567-570; Cramer, et al. (2001)Science 292, 1863-1876; Gnatt, et al. (2001) Science 292, 1876-1882).

Several residues of the switch region make direct contacts with DNAphosphates in the transcription elongation complex (Gnatt, et al. (2001)Science 292, 1876-1882; Westover, et al. (2004a) Science 303, 1014-1016;Westover, et al. (2004a) Science 303, 1014-1016; Westover, et al.(2004b) Cell 119, 481-489; Kettenberger, et al. (2004) Mol. Cell 16,955-965). Furthermore, it has been proposed that direct contents betweenthe switch region and DNA phosphates might coordinate, and even mightmechanically couple, clamp closure and DNA binding (Cramer, et al.(2002) Curr. Opin. Structl. Biol. 12 89-97; Landick, et al. (2001) Cell105, 567-570; Cramer, et al. (2001) Science 292, 1863-1876; and Gnatt,et al. (2001) Science 292, 1876-1882).

SUMMARY OF THE INVENTION

Applicant has discovered a target, located within the bacterial RNAPswitch region, through which small molecules are able to bind tobacterial RNAP, to inhibit bacterial RNAP, and to inhibit bacterialgrowth (FIGS. 2,3). The target comprises four short segments of thebacterial RNAP β′ and β subunits (FIG. 3). The target comprises residuesthat correspond to, and are alignable with, residues 345 and 1351 of theβ′ subunit of RNAP from Escerichia coli and residues 1275-1292 and1322-1326 of the β subunit of RNAP from Escerichia coli (FIG. 3).Throughout the following specification, said target is referred to asthe “switch-region target,” and said four short segments collectivelyare referred to as the “homologous switch-region amino-acid sequence.”

Applicant's results establish that the switch-region target is anexceptionally attractive target for drug discovery: (1) Theswitch-region target involves a critical structural element of bacterialRNAP (a structural element proposed to mediate conformation changesrequired for RNAP to bind to and retain the DNA template intranscription), providing a basis for efficient inhibition of bacterialRNAP and bacterial growth. (2) The switch-region target is conserved inGram-positive and Gram-negative bacterial RNAP, providing a basis forbroad-spectrum activity. (3) The switch-region target contains residuesthat are not conserved in human RNAP I, RNAP II, and RNAP III, providinga basis for therapeutic selectivity. (4) The switch-region target isremote from the binding site for the RNAP inhibitors in current clinicaluse (rifamycins), providing a basis for complete absence ofcross-resistance with the RNAP inhibitors in current clinical use. (5)Applicant has identified three natural products that bind to theswitch-region target, inhibit bacterial RNAP through the switch-regiontarget, and inhibit bacterial growth through the switch-regiontarget—providing examples of switch-region-target-dependent inhibitors.(6) Applicant and Applicant's co-workers have determined ahigh-resolution crystal structure of a complex of RNAP with one naturalproduct that binds to the switch-region target, inhibits bacterial RNAPthrough the switch-region target, and inhibits bacterial growth throughthe switch-region target—enabling structure-based screening for newswitch-region-target-dependent inhibitors. (7) Applicant has developedbinding, enzymatic-activity, and antibacterial-activity assays for smallmolecules that bind to the switch-region target, inhibit bacterial RNAPthrough the switch-region target, and inhibit bacterial growth throughthe switch-region target—enabling de novo screening for newswitch-region-target-dependent inhibitors.

Applicant has discovered that a sub-region within the bacterial RNAPswitch region comprising four short segments of the RNAP β′ and βsubunits is conserved in amino-acid sequence in bacterial species,including both Gram-positive bacterial species and Gram-negativebacterial species. The four short segments correspond to, and arealignable with, residues 345 and 1351 of the β′ subunit of RNAP fromEscerichia coli and residues 1275-1292 and 1322-1326 of the β subunit ofRNAP from Escerichia coli. Applicant further has discovered that thissub-region is not conserved, and in fact is radically different, inamino-acid sequence in eukaryotic RNAP, including human RNAP I, humanRNAP II, and human RNAP III. Applicant further has discovered that thistarget forms a discrete pocket, located in the center of the switchregion, in the three-dimensional structure of bacterial RNAP.

Accordingly, a first aspect of the present invention is directed to amethod for identifying agents that bind to a bacterial RNAP homologousswitch-region amino-acid sequence, comprising preparing a reactionsolution including the agent to be tested and an entity containing abacterial-RNAP homologous switch-region amino-acid sequence; anddetecting the presence or amount of binding to the bacterial-RNAPhomologous switch-region amino-acid sequence. In a preferred embodiment,detection or quantitation of binding is conducted relative to binding ofthe agent to an entity containing an altered bacterial-RNAP homologousswitch-region amino-acid sequence.

Another aspect of the present invention is directed to a method foridentifying agents that inhibit an activity of bacterial RNAP viabinding to a bacterial-RNAP homologous switch-region amino-acidsequence. This aspect entails preparing a reaction solution includingthe agent to be tested, an entity containing a bacterial-RNAP homologousswitch-region amino-acid sequence, and a substrate for the entity; anddetermining the extent of inhibition of an activity of the entity viabinding of the agent to the bacterial-RNAP homologous switch-regionamino-acid sequence. In a preferred embodiment, detection orquantitation of inhibition is conducted relative to inhibition by theagent of an entity containing an altered bacterial-RNAP homologousswitch-region amino-acid sequence.

Another aspect of the present invention is directed to a method foridentifying agents that inhibit at least one of bacterial viability andbacterial growth via binding to a bacterial-RNAP homologousswitch-region amino-acid sequence. This aspect entails contacting abacterium with the agent to be tested, and determining the extent ofinhibition of at least one of bacterial viability and bacterial growth.In a preferred embodiment, detection or quantitation of inhibition isconducted relative to inhibition by the agent of viability or growth ofa bacterium containing an altered bacterial-RNAP homologousswitch-region amino-acid sequence.

In some preferred embodiments, binding or inhibition is compared tobinding or inhibition by myxopyronin (Myx), corallopyronin (Cor), orripostatin (Rip). Applicant has discovered that each of these compoundsinhibits bacterial RNAP through interaction with the bacterial-RNAPhomologous switch-region amino-acid sequence. Applicant's resultsindicate that Myx, Cor, and Rip interact with residues that areconserved in Gram-positive and Gram-negative bacterial RNAP and,accordingly, exhibit broad-spectrum antibacterial activity. Applicant'sresults indicate that Myx, Cor, and Rip interact, in part, with residuesthat are not conserved in eukaryotic RNAP I, RNAP II, and RNAP III, and,accordingly, do not exhibit cross-inhibition of eukaryotic RNAP.Applicant's results further indicate that Myx, Cor, and Rip interactwith residues that are remote from the binding sites for rifamycins andother characterized RNAP inhibitors, and, accordingly, do not exhibitcross-resistance with rifamycins and other characterized RNAPinhibitors. Applicant's results further indicate that Myx, Cor, and Ripmay function by inhibiting switch-region conformational cycling, therebypreventing the opening of the RNAP clamp required for DNA binding and/orthe closing of the RNAP clamp required for DNA retention. Takentogether, these properties render Myx, Cor, and Rip exceptionallyattractive candidates for development as antibacterial therapeuticagents.

The present invention provides that each of Myx, Cor, and Rip inhibitsbacterial RNAP by binding to a determinant that includes residues withinthe bacterial RNAP homologous switch-region amino-acid sequence.

The present invention also provides for the identification of potentialantibacterial agents that, because they interact with residues that areconserved in bacterial RNAP, have broad-spectrum antibacterial activity.The invention also provides for the identification of potentialantibacterial agents that, because they interact, in part, with residuesthat are not conserved in eukaryotic RNAP, are relatively non-disruptiveto normal cellular functions of eukaryotes.

The invention also provides for the identification of potentialantibacterial agents that, because they interact with residues that areremote from the binding sites for rifamycins and other characterizedRNAP inhibitors, do not exhibit cross-resistance with rifamycins andother characterized RNAP inhibitors.

It is anticipated that compounds identified according to the target andmethod of this invention would have applications not only inantibacterial therapy, but also in: (a) identification of bacterial RNAP(diagnostics, environmental-monitoring, and sensors applications); (b)labeling of bacterial RNAP (diagnostics, environmental-monitoring,imaging, and sensors applications); (c) immobilization of bacterial RNAP(diagnostics, environmental-monitoring, and sensors applications); (d)purification of bacterial RNAP (biotechnology applications); (e)regulation of bacterial gene expression (biotechnology applications);and (f) antisepsis (antiseptics, disinfectants, and advanced-materialsapplications).

These and other aspects of the present invention will be betterappreciated by reference to the following drawings and DetailedDescription.

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the three-dimensional structure of bacterial RNAP (β′nonconserved domain and σ omitted for clarity). (A) Structure of RNAP,showing open (red), intermediate (yellow) and closed (green) clampconformations, as observed in crystal structures; violet sphere,active-center Mg²⁺. Two orthogonal views are shown: at left, a viewthrough the RNAP active-center cleft; at right, a view directly into theRNAP active-center cleft. (B) Structure of RNAP switch-region, showingopen (red), intermediate (yellow) and closed (green) clampconformations, as observed in crystal structures. A stereoview is shownof the structural elements known as “switch 2” and “switch 1” (β′residues 330-349 and 1304-1329; residues numbered as in Escerichia coliRNAP). Gray squares, points of connection of switch 2 and switch 1 tothe RNAP main mass; colored circles, points of connection of switch 2and switch 1 to the RNAP clamp.

FIG. 2 illustrates the location of the bacterial RNAP homologousswitch-region amino-acid sequence within the three-dimensional structureof bacterial RNAP (β′ nonconserved domain and σ omitted for clarity).Red, residues of the bacterial RNAP homologous switch-region amino-acidsequence; violet sphere, active-center Mg²⁺. Two orthogonal views areshown: at left, a view through the RNAP active-center cleft; at right, aview directly into the RNAP active-center cleft.

FIG. 3 shows sequence alignments of the bacterial RNAP homologousswitch-region amino-acid sequences (red boxes) from Escerichia coli,Haemophilus influenzae, Vibrio cholerae, Pseudomonas aeruginosa,Treponema pallidum, Borrelia burgdorferi, Xyella fastidiosa,Campylobacter jejuni, Neisseria meningitidis, Rickettsia prowazekii,Chlamydia trachomatis, Mycoplasma pneumoniae, Bacillus subtilis,Staphylococcus aureus, Mycobacterium tuberculosis, Synechocystis sp.,Aquifex aeolicus, Deinococcus radiodurans, Thermus thermophilus, andThermus aquaticus; and of the corresponding residues of human RNAP I,RNAP II, and RNAP III. Sequences for bacterial RNAP are at top;sequences for human RNAP I, RNAP II, and RNAP III are at bottom. Thesequence alignments include both the bacterial RNAP homologousswitch-region amino-acid sequences and the adjacent flanking sequences;the bacterial RNAP homologous switch-region amino-acid sequences areindicated by red boxes. (A) Sequences of β′ subunits of bacterial RNAPand largest subunits of human RNAP I, RNAP II, and RNAP III. (B)Sequences of β subunits of bacterial RNAP and second-largest subunits ofhuman RNAP I, RNAP II, and RNAP III.

FIG. 4 shows the chemical structures of myxopyronin (Myx),corallopyronin (Cor) and ripostatin (Rip).

FIG. 5 shows the locations of Myx-resistant substitutions within thethree-dimensional structure of bacterial RNAP (β′ nonconserved domainand σ omitted for clarity). Red, sites of single-residue substitutionsconferring high-level Myx-resistance; pink, sites of single-residuesubstitutions conferring moderate-level Myx-resistance; violet sphere,active-center Mg²⁺. Two orthogonal views are shown: at left, a viewthrough the RNAP active-center cleft; at right, a view directly into theRNAP active-center cleft. (A) Myx-resistant substitutions from randommutagenesis and selection. (B) Myx-resistant substitutions fromsaturation mutagenesis and selection.

FIG. 6 shows sequence alignments for segments of Escerichia coli β′subunit (A) and β subunit (B) in which single-residue substitutionsconferring Myx-resistance are obtained (sites of high-level resistanceboxed in red; sites of moderate-level resistance boxed in black).Sequences for bacterial RNAP are at top; sequences for human RNAP I,RNAP II, and RNAP III are at bottom.

FIG. 7 presents results indicating that Myx binds to Escerichia coliRNAP in a switch-region-target-dependent fashion (fluorescence quenchingequilibrium binding experiments; methods essentially as described inYarbrough, et al. (1976) J. Biol. Chem. 15, 2669-2676). ●, wild-typeRNAP; ◯, [Arg 345] β′-RNAP (RNAP derivative with single-residuesubstitution in bacterial RNAP homologous switch-region amino acidsequence).

FIG. 8 presents results indicating that Myx inhibits interaction ofEscerichia coli RNAP with promoter DNA in aswitch-region-target-dependent fashion (florescence-detectedelectrophoretic mobility shift assays; methods as in Mukhopadhyay, etal. (2004) Mol. Cell 14, 739-751). ●, wild-type RNAP; ◯, [Arg 345]β′-RNAP (RNAP derivative with single-residue substitution inswitch-region target). (A) DNA construct. (B) Data.

FIG. 9 presents results indicating that Myx does not inhibit interactionof Escerichia coli RNAP with the promoter DNA segment comprisingpositions −40 to −12 (fluorescence-detected electrophoretic mobilityshift assays; methods as in Mukhopadhyay, et al. (2004) Mol. Cell 14,739-751). (A) DNA construct. (B) Data.

FIG. 10 shows the crystal structure of the Thermus thermophilus RNAP-Myxcomplex. (A) Overall structure of the RNAP-Myx complex (β′ nonconserveddomain and σ omitted for clarity). Green, Myx; violet sphere,active-center Mg²⁺. Two orthogonal views: at leaft, view through theRNAP active-center cleft; at right, view directly into the RNAPactive-center cleft. (B) Stereoview showing details of interactionsbetween the bacterial RNAP homologous switch-region amino-acid sequenceand Myx. Blue, RNAP (ribbon representation of RNAP backbone atoms);green, Myx; red, sites of single-residue substitutions that conferhigh-level resistance to Myx.

BEST MODE OF CARRYING OUT THE INVENTION

The present invention provides methods of designing specific inhibitorsof bacterial RNAP, the enzyme responsible for transcription. Theinvention provides targets and methods for specific binding andinhibition of bacterial RNAP. The invention has applications in controlof bacterial gene expression, control of bacterial growth, antibacterialchemistry, and antibacterial therapy.

As described above, structural information for bacterial RNAP impliesthat the RNAP switch region serves as a hinge that permits rotation ofthe β′ subunit (termed the “clamp”) relative to the remainder of RNAP,and correspondingly, that permits opening or closing of the RNAPactive-center cleft (FIG. 1A). The clamp is proposed to open to permitentry of DNA into the active-center cleft in transcription initiation.The clamp is proposed to close to permit stable retention of DNA withinthe active-center cleft in later steps of transcription initiation andin transcription elongation. In addition to serving as the hinge for theproposed opening and closing of the clamp in transcription initiationand elongation, the switch region is proposed to make direct contactswith DNA in transcription initiation and in transcription elongation. Insummary, the switch region is proposed to play roles important forfunction of RNAP in transcription initiation and in transcriptionelongation.

It now has been found, and is disclosed herein, that binding of Myx,Cor, or Rip within the switch region inhibits transcription.Specifically, it has now been found, and is disclosed herein, thatbinding of Myx, Cor, or Rip within the switch region inhibitstranscription by preventing stable interaction of RNAP with apromoter-DNA segment that binds with the RNAP active-center cleft.

The present invention includes the discovery that a region within thebacterial RNAP switch region comprising residues corresponding to, andalignable with, residues 345 and 1351 of the β′ subunit of RNAP fromEscerichia coli and residues 1275-1292 and 1322-1326 of the β subunit ofRNAP from Escerichia coli (the “switch-region target” or “homologousswitch-region amino-acid sequence”; FIGS. 2,3) is a useful target forcompounds that block transcription. It was found that residues of thebacterial RNAP homologous switch-region amino-acid sequence areinvariant, or nearly invariant, in RNAP from bacterial species, but are,for at least in part, radically different in RNAP from eukaryoticspecies (FIG. 3). It further was found that, in the three-dimensionalstructure of bacterial RNAP, residues of the bacterial RNAP homologousswitch-region amino-acid sequence form a discrete pocket, withdimensions of approximately 20×20×10 Å, located within the RNAP switchregion (FIG. 2).

The location of the target within the bacterial RNAP switch region issuch that binding to the target of a small molecule would be predictedto lock the switch region in one conformation—either a conformation of aopen-clamp state, a conformation of an intermediate-clamp state, aconformation of a closed-clamp state, or an aberrant,small-molecule-dependent conformation. This would be predicted toprevent switch-region conformational cycling required for entry of DNAinto the active-center cleft, for stable binding of DNA within theactive-center cleft, or for both. The location of the target within theswitch region also is such that binding to the target of a smallmolecule might be predicted to interfere with interactions between RNAPand DNA (either by inhibiting transcription through allostericinteractions or through steric clash with the DNA template strand).

The target referred to above is highly similar in amino-acid sequence inRNAP from most or all other species of bacteria and is referred toherein as the “switch-region target” or the “homologous switch-regionamino-acid sequence” (FIGS. 2,3). (For example, residues 345 and 1351 ofthe β′ subunit and residues 1275-1292 and 1322-1326 of the β subunit ofRNAP from Escerichia coli correspond to, and are alignable with,residues 334 and 1165 of the β′ subunit and residues 1080-1097 and1127-1131 of the β subunit of RNAP from Bacillus subtilis; FIG. 3.)Thus, a molecule found to bind to the switch-region target and inhibitan activity associated with the switch-region target in RNAP from onespecies of bacteria, for example RNAP from Escerichia coli, is likelyalso to bind to the target and inhibit an activity associated with theswitch-region target in RNAP from other species of bacteria. Likewise, amolecule found to have antibacterial activity (through binding to andinhibiting an activity associated with the switch-region target) againstone species of bacteria, for example Escerichia coli, is likely to haveantibacterial activity against other species of bacteria.

In contrast, the target is not similar, and in part differs radically,in amino acid sequence between bacterial RNAP and eukaryotic RNAP,including human RNAP I, human RNAP II, and human RNAP III (FIG. 3). Thisallows for the identification of molecules that bind in aswitch-region-target-dependent fashion to bacterial RNAP, but that donot bind, or that bind substantially less well, to eukaryotic RNAP. Thisalso allows for the identification of molecules that inhibit in aswitch-region-target-dependent fashion an activity of bacterial RNAP,but that do not inhibit, or that inhibit substantially less well, anactivity of eukaryotic RNAP. This differentiation is important, becauseit permits the identification of bacterial-RNAP-selective bindingmolecules and bacterial-RNAP-selective inhibitors.

The invention provides, by way of example only, a target regioncorresponding to, and alignable with, residues 345 and 1351 of the β′subunit and residues 1275-1292 and 1322-1326 of the β subunit of RNAPfrom Escerichia coli, as well as corresponding residues of the β′ and βsubunits of RNAP from Haemophilus influenzae, Vibrio cholerae,Pseudomonas aeruginosa, Treponema pallidum, Borrelia burgdorferi, Xyellafastidiosa, Campylobacter jejuni, Neisseria meningitidis, Rickettsiaprowazekii, Thermotoga maritima, Chlamydia trachomatis, Mycoplasmapneumoniae, Bacillus subtilis, Staphylococcus aureus, Mycobacteriumtuberculosis, Synechocystis sp., Aquifex aeolicus, Deinococcusradiodurans, Thermus thermophilus, and Thermus aquaticus (FIG. 3). Thistarget region is the bacterial RNAP homologous switch-region amino-acidsequence.

The invention also provides compounds that bind to RNAP from a bacterialspecies, by making specific interactions with at least one residuewithin the set of residues corresponding to, and alignable with,residues 345 and 1351 of the β′ subunit and residues 1275-1292 and1322-1326 of the β subunit of RNAP from Escerichia coli.

The invention also provides compounds that inhibit an activity of RNAPfrom a bacterial species, by making specific interactions with at leastone residue within the set of residues corresponding to, and alignablewith, residues 345 and 1351 of the β′ subunit and residues 1275-1292 and1322-1326 of the β subunit of RNAP from Escerichia coli.

The invention also provides compounds that inhibit at least one ofviability of a bacterium and growth of a bacterium, by making specificinteractions with at least one residue within the set of residuescorresponding to, and alignable with, residues 345 and 1351 of the β′subunit and residues 1275-1292 and 1322-1326 of the β subunit of RNAPfrom Escerichia coli.

The invention provides identification of aswitch-region-target-dependent inhibitory compound by screening of achemical library for a molecule that: (a) binds to RNAP from a bacterialspecies, and (b) does not bind, or binds less well, to a derivative ofRNAP from a bacterial species that has at least one amino acidsubstitution, deletion, or insertion, in a bacterial RNAP homologousswitch-region amino-acid sequence.

The invention also provides identification of aswitch-region-target-dependent inhibitory compound by screening of achemical library for a molecule that: (a) inhibits enzymatic activity ofRNAP from a bacterial species, and (b) does not inhibit enzymaticactivity, or inhibits enzymatic activity less well, of a derivative ofRNAP from a bacterial species that has at least one amino acidsubstitution, deletion, or insertion, in a bacterial RNAP homologousswitch-region amino-acid sequence.

The invention also provides identification of aswitch-region-target-dependent inhibitory compound by screening achemical library for a molecule that: (a) inhibits DNA binding by RNAPfrom a bacterial species, and (b) does not inhibit DNA binding, orinhibits DNA binding less well, by a derivative of RNAP from a bacterialspecies that has at least one amino acid substitution, deletion, orinsertion, in a bacterial RNAP homologous switch-region amino-acidsequence.

The invention also provides identification of aswitch-region-target-dependent inhibitory compound by screening achemical library for a molecule that: (a) inhibits viability or growthof a bacterium, and (b) does not inhibit viability or growth, orinhibits viability or growth less well, of a bacterium that contains aderivative of RNAP from a bacterial species that has at least one aminoacid substitution, deletion, or insertion, in a bacterial RNAPhomologous switch-region amino-acid sequence.

The invention also provides identification of aswitch-region-target-dependent inhibitory compound by screening of achemical library for a first molecule that competes with a secondmolecule for binding to RNAP from a bacterial species, said secondmolecule having the ability to bind to a bacterial RNAP homologousswitch-region amino-acid sequence and containing a detectable group.

The invention also provides identification of aswitch-region-target-dependent inhibitory compound by use of at leastone of computational docking and energy calculations with a portion ofthe three-dimensional structure of a RNAP from a bacterial species, saidportion containing at least one residue of a bacterial RNAP homologousswitch-region amino-acid sequence.

The invention also provides for use of a molecule specific for abacterial RNAP homologous switch-region amino-acid sequence to identify,isolate, and/or immobilize RNAP from a bacterial species.

The invention also provides for use of a molecule specific for abacterial RNAP homologous switch-region amino-acid sequence to controlbacterial gene expression.

The invention also provides for use of a molecule specific for abacterial RNAP homologous switch-region amino-acid sequence to controlbacterial viability or bacterial growth.

The invention also provides for use of a molecule specific for abacterial RNAP homologous switch-region amino-acid sequence as anantibacterial agent.

One preferred aspect of the invention provides for a molecule specificfor a bacterial RNAP homologous switch-region amino-acid sequence thatbinds to RNAP from a bacterial species, but does not bind, or binds lesswell, to RNAP from a mammalian species.

Another preferred aspect of the invention provides for a moleculespecific for a bacterial RNAP homologous switch-region amino-acidsequence that inhibits biochemical activity of RNAP from a bacterialspecies, but does not inhibit biochemical activity, or inhibitsbiochemical activity less well, of RNAP from a mammalian species.

Another preferred aspect of the invention provides for a moleculespecific for a bacterial RNAP homologous switch-region amino-acidsequence that inhibits viability or growth of a bacterial species, butdoes not inhibit viability or growth, or inhibits viability or growthless well, of a mammalian species.

Another preferred aspect of the invention provides for a moleculespecific for a bacterial RNAP homologous switch-region amino-acidsequence that binds to and/or inhibits RNAP from a broad spectrum ofbacterial species.

Another preferred aspect of the invention provides for a moleculespecific for a bacterial RNAP homologous switch-region amino-acidsequence that binds to and/or inhibits RNAP from a broad spectrum ofGram-negative bacterial species.

Another preferred aspect of the invention provides for a moleculespecific for a bacterial RNAP homologous switch-region amino-acidsequence that binds to and/or inhibits RNAP from a broad spectrum ofGram-positive bacterial species.

Another preferred aspect of the invention provides for a moleculespecific for a bacterial RNAP homologous switch-region amino-acidsequence that binds to and/or inhibits RNAP from a broad spectrum ofboth Gram-negative and Gram-positive bacterial species.

Another preferred aspect of the invention provides for a molecule thatbinds to and/or inhibits RNAP from Escerichia coli, making specificinteractions with at least one residue within the set consisting ofresidues 345 and 1351 of the β′ subunit and residues 1275-1292 and1322-1326 of the β subunit of RNAP from Escerichia coli.

Another preferred aspect of the invention provides for a molecule thatbinds to and/or inhibits RNAP from Bacillus subtilis, making specificinteractions with at least one residue within the set consisting ofresidues 334 and 1165 of the β′ subunit and residues 1080-1097 and1127-1131 of the β subunit of RNAP from Bacillus subtilis.

The present invention further relates to a method for identifyingmolecules that bind to bacterial RNAP in aswitch-region-target-dependent fashion. In one embodiment, Escerichiacoli RNAP, or a fragment thereof, containing the switch-region target,is used as the test protein to assess binding, and a derivative of saidRNAP or RNAP fragment having at least one of a substitution, aninsertion, and a deletion within the switch-region target is used as thecontrol protein to assess switch-region-target-dependence of binding.“Hits” optionally may be analyzed for binding to, and inhibition of,Gram-negative-bacterial RNAP, Gram-positive-bacterial RNAP, andeukaryotic RNAP I, RNAP III and RNAP III, in vivo and in vitro. “Hits”optionally may also be characterized structurally by x-ray diffractionanalysis of co-crystals with RNAP or an RNAP fragment containing theswitch-region target.

The present invention further relates to a method for identifyingmolecules that inhibit an activity of a bacterial RNAP in aswitch-region-target-dependent fashion. In one embodiment, Escerichiacoli RNAP, or a fragment thereof, containing the switch-region target,is used as the test protein to assess inhibition, and a derivative ofsaid RNAP or RNAP fragment having at least one of a substitution, aninsertion, and a deletion within the switch-region target is used as thecontrol protein to assess switch-region-target-dependence of inhibition.“Hits” optionally may be analyzed for binding to, and inhibition of,Gram-negative-bacterial RNAP, Gram-positive-bacterial RNAP, andeukaryotic RNAP I, RNAP III and RNAP III, in vivo and in vitro. “Hits”optionally may also be characterized structurally by x-ray diffractionanalysis of co-crystals with RNAP or an RNAP fragment containing theswitch-region target.

The present invention further relates to a method for identifyingmolecules that inhibit viability and/or growth of a bacterium in aswitch-region-target-dependent fashion. In one embodiment, Escerichiacoli (preferably a tolC or tolC rfa strain of Escerichia coli; seeFralick, et al. (1994) J. Bacteriol. 176, 6404-6406) is used as the testbacterium to assess inhibition, and a derivative of Escerichia coli(preferably a tolC or tolC rfa strain of Escerichia coli; see Fralick,et al. (1994) J. Bacteriol. 176, 6404-6406) that contains an RNAPderivative having at least one of a substitution, an insertion, and adeletion within the switch-region target is used as the control toassess switch-region-target-dependence of inhibition. “Hits” optionallymay be analyzed for binding to, and inhibition of,Gram-negative-bacterial RNAP, Gram-positive-bacterial RNAP, andeukaryotic RNAP I, RNAP III and RNAP III, in vivo and in vitro. “Hits”optionally may also be characterized structurally by x-ray diffractionanalysis of co-crystals with RNAP or an RNAP fragment containing theswitch-region target.

The invention provides at least five assay methods for identification ofswitch-region-target-dependent inhibitors: a) screening based on bindingof a compound to the switch-region target of a bacterial RNAP or afragment thereof, b) screening based on inhibition of an activityassociated with the switch-region target of a bacterial RNAP or afragment thereof, c) screening based on inhibition of bacterialviability and/or growth dependent on the switch-region target of abacterial RNAP or a fragment thereof; d) screening based on competitionwith a second compound for binding to the switch-region target of abacterial RNAP or a fragment thereof, said second compound having theability to bind to the switch-region target and containing a detectablegroup; and e) computational screening using the three-dimensionalstructure of the switch-region target of a bacterial RNAP or a fragmentthereof.

One of the embodiments of the present invention is an assay systemdesigned to identify compounds that bind a bacterial RNAP, or a fragmentthereof, in a manner that requires the switch-region target. The assaymeasures the binding of a compound to a determinant that includes atleast one amino acid residue contained within a set of amino acidresidues identifiable by sequence alignment and/or structure alignmentas corresponding to, and alignable with, residues 345 and 1351 of the β′subunit and residues 1275-1292 and 1322-1326 of the β subunit of RNAPfrom Escerichia coli.

One of the embodiments of the present invention is an assay systemdesigned to identify compounds that inhibit an activity of a bacterialRNAP, or a fragment thereof, in a manner that requires the switch-regiontarget. The assay measures the inhibition of an activity, saidinhibition involving the binding of a compound to a determinant thatincludes at least one amino acid residue contained within a set of aminoacid residues identifiable by sequence alignment and/or structurealignment as corresponding to, and alignable with, residues 345 and 1351of the β′ subunit and residues 1275-1292 and 1322-1326 of the β subunitof RNAP from Escerichia coli.

One of the embodiments of the present invention is an assay systemdesigned to identify compounds that inhibit viability and/or growth of abacterium in a manner that requires the switch-region target. The assaymeasures the inhibition of viability and/or growth of a bacterium, saidinhibition involving the binding of a compound to a determinant thatincludes at least one amino acid residue contained within a set of aminoacid residues identifiable by sequence alignment and/or structurealignment as corresponding to, and alignable with, residues 345 and 1351of the β′ subunit and residues 1275-1292 and 1322-1326 of the β subunitof RNAP from Escerichia coli.

Isolation of RNAP:

The bacterial RNAP, or bacterial RNAP derivative, can be isolated frombacteria, produced by recombinant methods, or produced through in vitroprotein synthesis. Various compounds can be introduced to determinewhether a tested compound binds to, inhibits an activity of, ordisplaces a detectable-group-containing molecule from, the bacterialRNAP or RNAP derivative in a switch-region-target-dependent manner.

Assays can be performed in vitro or in vivo, and do not necessarilyrequire isolation of bacterial RNAP or bacterial RNAP derivative.

Test compounds can include peptides. Test compounds alternatively, or inaddition, can include non-peptide chemical compounds.

Test compounds can be chosen from chemical libraries. Test compoundsalternatively, or in addition, can be chosen based on informationregarding the three-dimensional structure of the switch-region target,using a computational approach, such as structure-based screening orstructure-based design.

Preferred strategies for identifying inhibitors include, but are notlimited to: 1) affinity-selection of phage-displayed peptide libraries,2) iterative deconvolution of solution-phase peptide libraries; 3)direct screening of solution-phase compound libraries; 4)structure-based screening; and 5) structure-based design. One ofEscerichia coli RNAP and Bacillus subtilis RNAP is the preferred testprotein to assess binding or inhibition of activity; one of a derivativeof Escerichia coli RNAP having at least one substitution in theswitch-region target and a derivative of Bacillus subtilis RNAP havingat least one substitution in the switch-region target is the preferredcontrol protein to assess switch-region-target dependence of binding orinhibition of activity. One of Escerichia coli (preferably a tolC ortolC rfa strain of Escherichia coli; see Fralick, et al. (1994) J.Bacteriol. 176, 6404-6406) and Bacillus subtilis is the preferred testbacterium to assess inhibition of viability and/or growth; one of aderivative of Escherichia coli (preferably a derivative of a tolC ortolC rfa strain of Escerichia coli; see Fralick, et al. (1994) J.Bacteriol. 176, 6404-6406) that contains a derivative of RNAP having atleast one substitution in the switch-region target and a derivative ofBacillus subtilis that contains a derivative of RNAP having at least onesubstitution in the switch-region target is the preferred controlbacterium to assess switch-region-target dependence of inhibition ofviability and/or growth.

Phage-Display Approach:

Millions to billions of short peptides readily can be surveyed for tightbinding to a protein target of interest by use of a phage-displayedpeptide library (Science 249:386; (1990) Science 249:404; and (1990)Proc. Natl. Acad. Sci. 87:6378). A phage-displayed peptide librarycomprises a mixture of filamentous phage clones, each displaying onespecific peptide sequence on the phage virion and each containing acorresponding nucleic-acid coding sequence in the phage virion. Thesurvey is accomplished by: (1) using the protein target of interest,typically immobilized on a surface or matrix, to affinity-purify thosephage that display tight-binding peptides; and (2) determiningnucleic-acid sequences of affinity-purified phage, thereby determiningencoded amino-acid sequences of tight-binding peptides. The surveytypically employs multiple successive cycles of affinity purification(with propagation of affinity-purified phage in a suitable bacterialhost between successive cycles) in order to ensure stringentaffinity-purification.

To identify peptides that bind to the switch-region target, aphage-displayed peptide library can be screened in two stages: a“positive-selection” stage and a “negative-selection” stage. In thepositive-selection stage, a bacterial RNAP or RNAP fragment, preferablyimmobilized on a surface or matrix, is used in at least one cycle ofaffinity-purification, collecting bound phage, in order to isolate thosephage that display peptides that bind tightly to any potential targetwithin the bacterial RNAP or RNAP fragment. In the negative-selectionstage, a derivative of a bacterial RNAP or RNAP fragment having asubstitution, insertion, or deletion within the switch-region target,preferably immobilized on a surface or matrix, is used in at least onecycle of affinity-purification, collecting unbound phage, in order toeliminate those phage that bind tightly to any potential targets otherthan the switch-region target within the bacterial RNAP or RNAPfragment.

Interative-Deconvolution and Positional-Scanning Approaches:

Iterative deconvolution (Houghten, et al. (1991) Nature 354, 84-86;Ostresh, et al. (2003) Meths. Enzymol. 267, 220-234; Hoesl, et al.(2003) Meths. Enzymol. 369, 496-517) and positional scanning (Ostresh,et al. (2003) Meths. Enzymol. 267, 220-234; Hoesl, et al. (2003) Meths.Enzymol. 369, 496-517) of solution-phase peptide libraries have beenestablished to be effective approaches to identifyreaction-step-specific, structural-element-specific inhibitors forstructure-function analysis in vitro (Puras, et al. (1995) Proc. Natl.Acad. Sci. USA 92, 11456-11460; Cassell, et al. (2000) J. Mol. Biol.299, 1193-1202; Klemm, et al. J. Mol. Biol. 299, 1203-1215; Boldt, etal. (2004) J. Biol. Chem. 279, 3472-3483), to identify antibacterialagents effective against cell-envelope targets (Houghten, et al. (1991)Nature 354, 84-86; Blondele, et al. (1996) Antimicrob. Agents Chemotehr.40, 1067-1071), and to identify antibacterial agents effective againstintracellular targets (Gunderson & Segall (2005) Mol. Microbiol. 59,1129-1148). Iterative deconvolution and positional scanning permiteffective screening of solution-phase tetrapeptide, pentapeptide,hexapeptide, and heptapeptide libraries comprising up to, respectively,160,000, 3,200,000, 64,000,000, and 1,280,000,000 distinct sequences ofstandard L-amino acids (Houghten, et al. (1991) Nature 354, 84-86;Ostresh, et al. (2003) Meths. Enzymol. 267, 220-234; Hoesl, et al.(2003) Meths. Enzymol. 369, 496-517). Iterative deconvolution andpositional scanning also permit effective screening of solution-phasepeptide libraries containing nonstandard amino acids, D-amino acids, andterminal or internal modifications (Houghten, et al. (1991) Nature 354,84-86; Ostresh, et al. (2003) Meths. Enzymol. 267, 220-234; Hoesl, etal. (2003) Meths. Enzymol. 369, 496-517).

Iterative deconvolution and positional scanning approaches employinitial solution-phase peptide libraries organized into pools, each poolcomprising multiple distinct peptide sequences (Houghten, et al. (1991)Nature 354, 84-86; Ostresh, et al. (2003) Meths. Enzymol. 267, 220-234;Hoesl, et al. (2003) Meths. Enzymol. 369, 496-517). The initial libraryis screened using an assay of interest, pools exhibiting activity arechosen for further analysis, and successive cycles of synthesis andscreening of subdivided pools are employed in order to identifyindividual peptides exhibiting activity (Houghten, et al. (1991) Nature354, 84-86; Ostresh, et al. (2003) Meths. Enzymol. 267, 220-234; Hoesl,et al. (2003) Meths. Enzymol. 369, 496-517).

To identify peptides that bind to the switch-region target, iterativedeconvolution or positional scanning of a solution-phase tetrapeptide,pentapeptide, hexapeptide, and heptapeptide hexapeptide or heptapeptidelibrary can be performed. In a preferred embodiment, iterativedeconvolution or positional scanning of a solution-phase pentapeptide,hexapeptide, or heptapeptide library is performed. (There is anapproximate agreement between: (a) molecular dimensions ofpentapeptides, hexapeptides, and heptapeptides; and (b) moleculardimensions of the switch-region-target ligands disclosed herein, Myx,Cor, and Rip.) Requisite screening steps can be performed using any oneor more of the assays described below for switch-region-target-dependentbinding, switch-region-target-dependent inhibition of activity, orswitch-region-target-dependent inhibition of bacterial viability orgrowth. In a preferred embodiment, requisite screening steps areperformed using a high-throughput assay forswitch-region-target-dependent inhibition of activity (see, e.g.,Example 4).

Direct Screening Approach:

Chemical libraries containing up to hundreds of thousands of singlecompounds can be directly screened, compound by compound, for binding toa protein of interest, for inhibition of an activity of a protein ofinterest, and/or for inhibition of viability or growth of a bacterium.In a preferred embodiment, high-throughput screening is performed, usingany one or more of the assays described below forswitch-region-target-dependent binding, switch-region-target-dependentinhibition of activity, or switch-region-target-dependent inhibition ofbacterial viability or growth. In an especially preferred embodiment,high-throughput screening is performed using an assay forswitch-region-target-dependent inhibition of activity (see, e.g.,Example 4).

High-throughput screening typically employs assays carried out in 96-,384- or 1536-well plates, according to methods well established in theart (see, e.g., Example 4). High-throughput screening can be performedby an individual laboratory or by a dedicated high-throughput screeningfacility (for example, the National Screening Laboratory for theRegional Centers of Excellence for Biodefense and Emerging InfectiousDisease, NSRB, which has access to over 160,000 compounds and whichtypically performs screens of 50,000-100,000 compounds;http:/nsrb.med.harvard.edu/).

Structure-Based-Screening Approach:

Structure-based screening permits analysis of higher numbers ofcompounds and higher numbers of distinct chemotypes than directscreening alone, and, as such, can permit analysis of a larger, morediverse, fraction of chemical space than direct screening alone(Muchmore & Hajduk (2003) Curr. Opin. Drug Discov. Dev. 6, 544-549;Alvarez, J. (2004) Curr. Opin. Chem. Biol. 8, 365-370; Shoichet, B.(2004) Nature 432, 862-865; Jorgensen, W. (2004) Science 303,1813-1818). Structure-based screening typically entails two stages. Inthe first stage, virtual screening of a large library of compounds(e.g., 100,000-1,000,000 compounds) is performed in order to identifycandidate compounds for further analysis; in this stage, for eachcompound, computational docking of the compound to the binding site ofinterest is carried out, binding free energy is estimated, and a scoreis assigned. In the second stage, confirmatory direct screening of asmall number of highest-scoring candidate compounds (e.g., 10-100compounds) is performed in order to validate and re-rank candidatecompounds.

The first stage, entailing virtual screening, can be performed by use ofvirtual-screening software packages, including, but not limited to,Glide, GOLD, ICM, LigandFit, FlexX, and DOCK (see Perola, et al. (2004)Proteins 56, 235-249; Kellenberger, et al. (2004) Proteins 57, 225-242;Kontoyianni, et al. (2004) J. Med. Chem. 47, 558-565; Chen, et al.(2006) J. Chem. Info Model. 46, 401-415). In a preferred embodiment,enhancements that allow for consideration of binding-site flexibilityand that thereby improve scoring accuracy, can be incorporated (see,e.g., Sherman, et al. (2006) J. Med. Chem. 49, 534-553). In a preferredembodiment, virtual screening employs a virtual compound librarycomprising structures of at least 1,000,000 purchasable compounds (e.g.,the virtual; library available through Schrödinger, Inc.) and employs atleast one structural template for the switch-region target, saidstructural template being the three-dimensional structure of theswitch-region target as in a crystal structure of unliganded bacterialRNAP or the three-dimensional structure of the switch-region target asin a crystal structure of a complex of bacterial RNAP with aswitch-region-target-dependent inhibitor. The use of multiple, differentstructural templates in parallel (e.g., the three-dimensional structureof the switch-region target as in a crystal structure of unligandedbacterial RNAP and the three-dimensional structure of the switch-regiontarget as in a crystal structure of a complex of bacterial RNAP with aswitch-region-target-dependent inhibitor) is especially preferred, sincethe local conformation of the switch-region target—including thedimensions, volume, shape, and chemical character of the pocket thatserves the binding site for potential inhibitors—can differ fordifferent structural templates, and, correspondingly, the universe ofpotential inhibitors can differ for different structural templates.

The second stage, entailing confirmatory direct screening, can beperformed using any one or more of the assays described below forswitch-region-target-dependent binding, switch-region-target-dependentinhibition of activity, or switch-region-target-dependent inhibition ofbacterial viability or growth. In a preferred embodiment, confirmatorydirect screening is performed assessing approximately 100 highest-rankedpurchasable candidate compounds for each structural template, and isperformed in high-throughput format using an assay forswitch-region-target-dependent inhibition of activity (see, e.g.,Example 4).

Structure-Based-Design Approach:

Starting with the three-dimensional structure of a switch-region target,or of a complex of a switch-region target and aswitch-region-target-dependent ligand, potential ligands for the targetcan be examined through the use of computational modeling using adocking program, such as Glide, GOLD, ICM, LigandFit, FlexX, or DOCK(see Perola, et al. (2004) Proteins 56, 235-249; Kellenberger, et al.(2004) Proteins 57, 225-242; Kontoyianni, et al. (2004) J. Med. Chem.47, 558-565; Chen, et al. (2006) J. Chem. Info. Model. 46, 401-415).This procedure can include computer fitting of potential ligands to theswitch-region target to ascertain the degree of compatibility betweenthe shape and the chemical structure of the potential ligand and theshape and chemical structure of the switch-region target. Computationalmethods also be can employed to estimate the attraction, repulsion, andsteric hindrance of a potential ligand with the switch-region target.

Known ligands of the switch-region target can be systematically modifiedby computer modeling programs until one or more promising potential newligands are identified. In addition, promising potential new ligandsligands can be systematically modified by computer modeling programsuntil one or more next-generation promising potential new ligands areidentified. This approach has been shown to be effective in thedevelopment of HIV protease inhibitors (Lam et al., Science 263:380-384(1994); Wlodawer et al., Ann. Rev. Biochem. 62, 543-585 (1993); Appelt,(1993) Perspectives in Drug Discovery and Design 1, 23-48; Erickson(1993) Perspectives in Drug Discovery and Design 1, 109-128).

Once a potential new ligand of the switch-region target is identified,it may be obtained from libraries of chemicals as are available frommost large chemical companies including Merck, Glaxo Welcome, BristolMeyers Squibb, Monsanto, Novartis, and Pfizer, or, alternatively, it maybe synthesized. The synthesis of one compound, or even a group ofcompounds, is reasonable in the art of drug design. The potential newligand then may be subjected to confirmatory direct screening, performedusing any one or more of the assays described below forswitch-region-target-dependent binding, switch-region-target-dependentinhibition of activity, or switch-region-target-dependent inhibition ofbacterial viability or growth.

When a new ligand of the switch-region-target is identified—by astructure-based design approach or by any of the above-describedapproaches—a crystal can be obtained of a complex of a bacterial RNAP orRNAP fragment and the new ligand, either by soaking or by de novocrystallization, and a crystal structure can be determined of saidcomplex. Preferably, the crystal can effectively diffract x-rays for thedetermination of the atomic coordinates of said complex to a resolutionof better than 4.0 Å. The crystal structure can be determined bymolecular replacement. Molecular replacement involves using a knownthree-dimensional structure, in this case the three-dimensionalstructure of unliganded bacterial RNAP or RNAP fragment, as a searchmodel to determine the structure of a closely related molecule orprotein-ligand complex in a new crystal form. The measured x-raydiffraction properties of the new crystal are compared with the searchmodel structure to compute the position and orientation of the proteinin the new crystal. Computer programs that can be used include: X-PLOR,CNS, (Crystallography and NMR System, a next level of XPLOR), and AMORE(J. Navaza, Acta Crystallographics ASO, 157-163 (1994)). Once theposition and orientation are known, an electron density map can becalculated using the search model to provide X-ray phases. Thereafter,the electron density can be inspected for structural differences and thesearch model can be modified to conform to the new structure.

Assay Components:

The bacterial RNAP, or RNAP fragment or derivative, containing theswitch-region target, and an inhibitory compound specific to the switchregion of RNAP, which are binding partners used as components in theassay, may be derived from natural sources (e.g., purified frombacterial RNAP using protein separation techniques well known in theart); produced by recombinant DNA technology using techniques known inthe art (see, e.g., Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratories Press, Cold SpringHarbor, N.Y.); and/or chemically synthesized in whole or in part usingtechniques known in the art (see, e.g., Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y.,pp.50-60).

Where recombinant DNA technology is used to produce the bacterial RNAP,RNAP fragment, or derivative containing the switch-region target, it maybe advantageous to engineer fusion proteins that can facilitatelabeling, immobilization and/or detection. For example, the codingsequence of a bacterial RNAP switch region can be fused to that of aheterologous protein that has enzyme activity or serves as an enzymesubstrate in order to facilitate labeling and detection. The fusionconstructs should be designed so that the heterologous component of thefusion product does not interfere with binding of the bacterial RNAPswitch region and an inhibitory compound specific to the switch regionof RNAP.

For a binding assay, one of the binding partners used in the assaysystem may be labeled, either directly or indirectly, to facilitatedetection of a complex formed between the bacterial RNAP switch regionand an inhibitory compound specific to the switch-region target of RNAP.Any of a variety of suitable labeling systems may be used including, butnot limited to, radioisotopes such as ¹²⁵I; enzyme labeling systems thatgenerate a detectable calorimetric signal or light when exposed tosubstrate; and fluorescent labels.

Fluorescent labels are preferred.

Indirect labeling involves the use of a third protein, such as a labeledantibody, which specifically binds to an entity containing aswitch-region target. Such antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, humanized, single chain, Fab fragmentsand fragments produced by an Fab expression library.

For the production of antibodies, various host animals may be immunizedby injection with at least one segment of an entity containing aswitch-region target. Such host animals may include, but are not limitedto, rabbits, mice, and rats, to name but a few. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

Monoclonal antibodies may be prepared by using any technique thatprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique originally described by Kohler and Milstein, (1975) Nature256:495-497), the human B-cell hybridoma technique (Kosbor et al. (1983)Immunology Today, 4:72, Cote et al. (1983) Proc. Natl. Acad. Sci.,80:2026-2030) and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). In addition, techniques developed for the production of“chimeric antibodies” (Morrison et al. (1984) Proc. Natl. Acad. Sci.,81:6851-6855; Neuberger et al. (1984) Nature, 312:604-608; Takeda et al.(1985) Nature, 314:452-454) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity.Alternatively, techniques described for the production of single-chainantibodies (e.g., U.S. Pat. No. 4,946,778) can be adapted to producesingle-chain antibodies specific to an entity containing a switch-regiontarget.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, such fragments include, but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.(1989) Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Binding Assays:

Binding assays can be conducted in a heterogeneous or homogeneousformat. A heterogeneous assay is an assay in which reaction results aremonitored by separating and detecting at least one component during orfollowing reaction. A homogeneous assay is an assay in which reactionresults are monitored without separation of components.

In either approach, the order of addition of reactants can be varied toobtain different information about the compounds being tested.

In one example of a heterogeneous binding assay system, one bindingpartner—e.g., either an entity containing a switch-region target or acompound specific to the switch-region target—is anchored onto a solidsurface, and the other binding partner, which is not anchored, islabeled, either directly or indirectly. In practice, microtiter platesare conveniently utilized. The anchored species may be immobilized bynon-covalent or covalent attachments. Alternatively, an immobilizedantibody specific for the an entity containing a switch-region targetmay be used to anchor the entity to the solid surface. The surfaces maybe prepared in advance and stored. In order to conduct the assay, thenon-immobilized binding partner is added to the coated surface with orwithout the test compound. After the reaction is complete, unreactedcomponents are removed (e.g., by washing) and any complexes formed willremain immobilized on the solid surface. The detection of complexesanchored on the solid surface can be accomplished in a number of ways.Where the binding partner was pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe binding partner is not pre-labeled, an indirect label can be used todetect complexes anchored on the surface; e.g., using a labeled antibodyspecific for the binding partner (the antibody, in turn, may be directlylabeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

In a preferred embodiment of the invention, a homogeneous binding assayis used. In one preferred embodiment of the invention, involving use ofa homogeneous binding assay, a preformed complex of an entity containinga switch-region target and a compound that binds to the switch-regiontarget is prepared, in which at least one of the binding partnerscontains a detectable group having that exhibits a difference in adetectable property in the complex state and in the free state (see,e.g., U.S. Pat. No. 4,109,496); the addition of a test compound thatcompetes with, and displaces, one of the binding partners from thepreformed complex results in a change in a detectable properties of thedetectable group, permitting identification of test substances able tobind to the switch-region target.

One aspect of the invention provides fluorescence resonance energytransfer (FRET)-based homogeneous assays (Förster (1948) Ann. Physik.(Leipzig) 2, 55-75; reviewed in Lilley and Wilson (2000) Curr. Opin.Chem. Biol. 4, 507-517; Selvin, P (2000) Nature Structl. Biol. 7,730-734; Mukhopadhyay et al., 2001 Cell 106, 453-463; Mekler, et al.(2002) Cell 108, 599-614; Mukhopadhyay, et al. (2004) Mol. Cell 14,739-751). FRET occurs in a system having a first fluorescent probeserving as a donor and a second fluorescent probe or chhomophore servingas an acceptor, where the emission wavelength of the donor overlaps theexcitation wavelength of the acceptor. In such a system, upon excitationof the donor with light of its excitation wavelength, energy can betransferred from the donor to the acceptor, resulting in excitation ofthe acceptor and omission at the acceptor's emission wavelength. Withcommonly used fluorescent probes, FRET permits accurate determination ofdistances in the range of ˜20 to ˜100 Å. FRET permits accuratedetermination of distances up to more than one-half the diameter of atranscription complex (diameter ˜150 Å; see Zhang et al. 1999; Cramer etal. (2001) Science 292, 1863-1876; Gnatt et al. (2001) Science 292,1876-1882).

A preferred assay involves monitoring of FRET between: a) one of afluorescent probe or a chromophore present in a bacterial RNAP, and b)one of a fluorescent probe or a chromophore present in a small moleculethat binds to the switch-region target.

An especially preferred assay involves monitoring of FRET between: a)one of a fluorescent probe or a chromophore present in a bacterial RNAP,and b) one of a fluorescent probe or a chromophore present in one ofMyx, Cor, or Rip (see, e.g., Example 1, section 1c).

Activity Assays:

In a particular embodiment, the effect of a test compound on an activityof a bacterial RNAP, or a fragment thereof, is determined (eitherindependently of, or subsequent to, a binding assay as exemplifiedabove). In one such embodiment, the extent or rate of the DNA-dependentRNA synthesis is determined. For such assays, a labeled nucleotide canbe used. The assay can include the withdrawal of aliquots from theincubation mixture at defined intervals and subsequent analysis.Alternatively, the assay can be performed using a real-time assay (e.g.,with a fluorescently labeled nucleotide or with a fluorescent probe forRNA).

One assay for RNAP activity is a modification of the method of Burgesset al. (J. Biol. Chem., 244:6160 (1969); seehttp://www.worthington-biochem.com/manual/R/RNAP.html). One unitincorporates one nanomole of UMP into acid insoluble products in 10minutes at 37° C. under the assay conditions such as those listed below.The suggested assay conditions are: (a) 0.04 M Tris-HCl, pH 7.9,containing 0.01 M MgCl₂, 0.15 M KCl, and 0.5 mg/ml BSA; (b) nucleosidetriphosphates (NTP): 0.15 mM each of ATP, CTP, GTP, UTP; spiked with³H-UTP 75000-150000 cpms/0.1 ml; (c) 0.15 mg/ml calf thymus DNA; (d) 10%cold perchloric acid; and (e) 1% cold perchloric acid. A starting enzymeconcentration of 0.1-0.5 units of RNAP in 5 μl-10 μl are used as thestarting enzyme concentration.

The procedure is to add 0.1 ml Tris-HCl, 0.1 ml NTP and 0.1 ml DNA to atest tube for each sample or blank. At time zero, enzyme (or buffer forblank) is added to each test tube, and the contents are then mixed andincubated at 37° C. for 10 minutes. 1 ml of 10% perchloric acid is addedto the tubes to stop the reaction. The acid insoluble products can becollected by vacuum filtration through Millipore filter discs having apore size of 0.45 u-10 u (or equivalent). The filters are then washedfour times with 1% cold perchloric acid using 1 ml-3 ml for each wash.These filters are then placed in scintillation vials. Two ml of methylcellosolve are added to the scintillation vials to dissolve the filters.When the filters are completely dissolved (after about five minutes) 10ml of scintillation fluid are added and the vials are counted in ascintillation counter.

Additional assays for analysis of RNAP activity contemplated by thepresent invention include fluorescence-detected abortive initiationassays, fluorescence-detected transcription assays, andmolecular-beacon-based transcription assays. An especially preferredassay is the fluorescence-detected abortive initiation assay (seeExample 4).

In assays of RNAP activity, different orders of addition of componentsmay be employed. In preferred embodiments, an order of addition isemployed in which RNAP or RNAP derivative is pre-incubated with the testcompound—affording time and opportunity for formation of a complexbetween RNAP or RNAP derivative and the test compound—before RNAP isincubated with DNA.

Antibacterial Assays:

Methods of testing a compound for antibacterial activity in cultures arewell known in the art, and can include standard assays of minimuminhibitory concentration (MIC; see, e.g., Examples 1-3, Tables 1 and2-6) and of minimum bacteriocidal concentration (MBC).

Animal Model Assays:

Inhibitors of bacterial RNAP identified by the processes of the presentinvention can be assayed in animal experiments. The ability of aninhibitor to control bacterial infection can be assayed in animal modelsthat are natural hosts for the bacterial species of interest. Suchanimal models may involve mammals, such as rodents, dogs, pigs, horses,and primates. Such animal models can be used to determine the LD₅₀ andthe ED₅₀ in animal subjects, and such data can be used to derive thetherapeutic index for the inhibitor. In animal models, test compoundscan be administered by a variety of routes including topical, oral,subcutaneous, and intraperitoneal routes, depending on the proposed use.Generally, at least two groups of animals are used in the assay, with atleast one group being a control group, which is administered theadministration vehicle without the test compound.

Pharmaceutical Preparations and Methods of Administration:

Identified compounds that inhibit bacterial replication can beadministered to a patient at therapeutically effective doses to treatbacterial infection. A therapeutically effective dose refers to thatamount of the compound sufficient to result in amelioration of symptomsof bacterial infection.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of infection in order to minimize damage to uninfected cells andreduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal infection, or ahalf-maximal inhibition) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates may be formulated for administration by inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebuliser, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycollate); or wetting agents (e.g., sodium lauryl sulphate).The tablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

DEFINITIONS

As used herein a “small molecule” is a compound that has a molecularweight of less than approximately 15 kDa.

As used herein a “small organic molecule” is an organic compound [ororganic compound complexed with an inorganic compound (e.g., metal)]that has a molecular weight of less than approximately 3 kDa.

As used herein the term “about” preferably means within 10 to 15%,preferably within 5 to 10%. For example, an amino acid sequence thatcontains about 60 amino acid residues preferably contains between 51 to69 amino acid residues, more preferably 57 to 63 amino acid residues.

As used herein the term “switch-region target” comprises amino acidresidues corresponding to, and alignable with, residues 345 and 1351 ofthe β′ subunit and residues 1275-1292 and 1322-1326 of the β subunit ofRNAP from Escerichia coli, or a set of residues corresponding to, andalignable with residues 334 and 1165 of the β′ subunit and residues1080-1097 and 1127-1131 of the β subunit of RNAP from Bacillus subtilis(FIGS. 2,3).

As used herein the term “homologous switch-region amino-acids sequence”comprises amino acid residues corresponding to, and alignable with,residues 345 and 1351 of the β′ subunit and residues 1275-1292 and1322-1326 of the β subunit of RNAP from Escerichia coli, or a set ofresidues corresponding to, and alignable with residues 334 and 1165 ofthe β′ subunit and residues 1080-1097 and 1127-1131 of the β subunit ofRNAP from Bacillus subtilis (FIGS. 2,3).

As used herein, the term “sequence homology” in all its grammaticalforms refers to the relationship between proteins that possess a “commonevolutionary origin,” including proteins from superfamilies (e.g., theimmunoglobulin superfamily) and homologous proteins from differentspecies (e.g., myosin light chain, etc) (Reeck et al. (1987) Cell 50,667).

Accordingly, the term “sequence similarity” in all its grammatical formsrefers to the degree of identity or correspondence between nucleic acidor amino acid sequences of proteins that do not share a commonevolutionary origin (see Reeck et al., 1987, supra). However, in commonusage and in the instant application, the term “homologous” may refer tosequence similarity and not a common evolutionary origin.

The term “corresponding to” is used herein to refer to similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases.

The present invention contemplates isolation of nucleic acids encodingthe target. The present invention further provides for subsequentmodification of the nucleic acid to generate a fragment or modificationof the target.

The present invention is not to be limited in scope by the specificembodiments describe herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

EXAMPLES

With reference to the examples below, Applicant has identified threecompounds that inhibit bacterial RNAP through interactions with the RNAPswitch region and that appear to function by preventing clamp openingrequired for DNA binding and/or clamp closure required for DNAretention: myxopyronin (Myx), corallopyronin (Cor), and ripostatin(Rip). The structures of these compounds are shown in FIG. 4. The threecompounds interact with residues that are conserved in Gram-positive andGram-negative bacterial RNAP, and, accordingly, exhibit broad-spectrumantibacterial activity. The three compounds interact, in part, withresidues that are not conserved in eukaryotic RNAP I, RNAP II, and RNAPIII, and, accordingly, do not exhibit cross-inhibition of eukaryoticRNAP. The three compounds interact with residues that are remote fromthe binding site for the rifamycins and from the binding sites for othercharacterized RNAP inhibitors, and, accordingly, do not exhibitcross-resistance with rifamycins or other characterized RNAP inhibitors.Taken together, these properties render the three compoundsexceptionally attractive candidates for development as antibacterialtherapeutic agents.

Example 1 Switch-Region-Target Inhibitors: Myx

The present example is directed to the use of myxopyronin (Myx) as asmall-molecule inhibitor of bacterial RNAP that, based on Applicant'sdiscovery, functions through interaction with the bacterial RNAPhomologous amino-acid sequence.

Myx is a polyketide-derived α-pyrone antibiotic (Irschik, et al. (1983)J. Antibiot 36, 1651-1658; Kohl, et al. (1983) Liebigs Ann. Chem.1656-1667; Kohl, et al. (1984) Liebigs Ann. Chem. 1088-1093; FIG. 4).Myx is produced by Myxococcus fulvus strain Mxf50 (DSM 2549; Irschik, etal. (1983) J. Antibiot 36, 1651-1658). The compound inhibits growth ofGram-positive and Gram-negative bacterial species, including Bacillussubtilis, B. megaterium, Staphylococcus aureus, Micrococcus luteus,Enterococcus faecium, Enterobacter cloacae, Corynebacterium mediolanum,Mycobacterium smegmatis, Acinetobacter calcoaceticus, Pseudomonasaeruginosa, and Escerichia coli DH21tolC (MICs≦10 μg/ml for all; MICs≦1μg/ml for S. aureus, A. calcoaceticus, and Escerichia coli DH21tolC ;Irschik, et al. (1983) Supra; Kohl, et al. (1983) Supra; unpublisheddata). The compound is bacteriocidal, as assessed with Escerichia coliDH21tolC (unpublished data). The compound inhibits bacterial RNAP(K_(i)=1 μM) but does not inhibit eukaryotic RNAP II (Irschik, et al.(1983) Supra; unpublished data). The compound exhibits no acute toxicityin mice at concentrations up to 100 mg/kg (Irschik, et al. (1983)Supra). The compound exhibits no cross-resistance with rifamycins,CBR-703, or microcin J25 (Hu, et al. (1988) Supra; unpublished data).Two total syntheses of racemic Myx have been reported (Hu, et al. (1988)J. Org. Chem. 63, 2401-2406; Doundoulakis, et al. (2004) Bioorg. Med.Chem. Lett. 14, 5667-5672).

1. Myx: Target of Transcription Inhibition

a. Myx Requires the Switch-Region Target: Results of Random Mutagenesisand Selection.

To identify determinants for function of Myx, Applicant has performedrandom mutagenesis of genes encoding Escerichia coli RNAP β′ subunit(rpoC) and β subunit (rpoB), and has isolated and sequenced fiveindependent Myx-resistant mutants (methods as described in Mukhopadhyay,et al. (2004) Mol. Cell 14, 739-751).

Results are presented in Table 1 and FIG. 5A. Substitutions conferringMyx-resistance were obtained at β′ residue 345 (three isolates) and at βresidues 1275 (one isolate) and 1292 (one isolate) (Table 1). In thethree-dimensional structure of bacterial RNAP, the sites of theMyx-resistant substitutions identified by random mutagenesis andselection define a discrete, continuous determinant with dimensions ofapproximately 10×10×5 Å (FIG. 5A). The determinant is located within thebacterial RNAP switch region and, in particular, is located within thebacterial RNAP homologous switch-region amino-acid sequence (FIG. 5A;compare FIG. 2). All substitutions conferring Myx-resistance affectresidues of the bacterial RNAP homologous switch-region amino-acidsequence (compare FIG. 3). The results establish that inhibition oftranscription by Myx requires the bacterial RNAP switch-regionhomologous amino-acid sequence. TABLE 1 Myx^(r) isolates from randommutagenesis and selection number of amino acid codon independent MICsubstitution substitution isolates ratio rpoC  345 Lys→Asn AAG→AAT  2*32  345 Lys→Thr AAG→ACG 1 32 rpoB 1275 Val→Met GTG→ATG  1** >32 1291Leu→Phe CTC→TTC 1 2*One isolate obtained as double mutant 340 Gln→Leu; 345 Lys→Asn.**Isolated as double mutant 857 Val→Met; 1275 Val→Met; phenotypeconfirmed as single mutant constructed by site-directed mutagenesisb. Myx Requires the Switch-Region Target: Results of SaturationMutagenesis and Selection.

To further define determinants for function of Myx, Applicant hasperformed saturation mutagenesis of genes encoding Escerichia coli RNAPβ′ subunit (rpoc) and β subunit (rpoB), and has isolated and sequencedmore than 100 independent Myx-resistant mutants (methods as described inMukhopadhyay, et al. (2004) Mol. Cell 14, 739-751; Tuske, et al. (205)Cell 122, 541-552). Saturation mutagenesis was performed using a set of“doped” oligodeoxribonucleotide primers targeting all codons forresidues located within 30 Å of β′ residue 345 and β residues 1275 and1292 in the three-dimensional structure of bacterial RNAP (primersequences in Table 2).

Results are presented in Table 3, FIG. 5B, and FIG. 6. Single-residuesubstitutions conferring Myx-resistance were obtained at β′ residues 345and 1351, and at β residues 1255, 1275, 1278, 1279, 1285, 1298, 1315,1317, 1320, 1322, and 1325 (Table 3). In the three-dimensional structureof bacterial RNAP, the sites of the Myx-resistant substitutions define adeterminant with dimensions of approximately 20×20×10 Å (FIG. 5B). Thedeterminant is located within the bacterial RNAP switch region and, inparticular, is located within the bacterial RNAP homologousswitch-region amino-acid sequence (FIG. 5B; compare FIG. 2). All fivehigh-level Myx-resistant substitutions affect residues of the bacterialRNAP homologous switch-region amino-acid sequence (β′ residues 345 and1351 and β residues 1275, 1279, and 1322; FIG. 6; compare FIG. 3). Fourof five high-level Myx-resistant substitutions affect residues that areconserved in bacterial RNAP but that are not conserved in eukaryoticRNAP I, RNAP II, or RNAP III, consistent with the selectivity of Myx (β′residue 1351 and β residues 1275, 1279, and 1322; FIG. 6). The resultsestablish that inhibition of transcription by Myx requires the bacterialRNAP switch-region homologous amino-acid sequence. TABLE 2“doped” oligonucleotide primers used in saturation mutagenesis codonstargeted sequence rpoC 325-335 GCGTCCTCTGAAATCTTTGGCCGACATGATCAAAGGTAAACAGGGTCGTTTCCG 336-346 GGTAAACAGGGTCGTTTCCGTCAGAACCTGCTCGGTAAGCGTGTTGACTACTCC 347-355 CGGTAAGCGTGTTGACTACTCCGGTCGTTCTGTAATCACCG TAGGTC429-433 TGCACCGACTCTGCACCGTCTGGGTATCCAGGCAT 466-481GGTGACCAGATGGCTGTTCACGTACCGCTGACGCTGGAAGC CCAGCTGGAAGCGCGTGCGCTGATG794-807 GCGAACTCCGGTTACCTGACTCGTCGTCTGGTTGACGTGGC GCAGGACCTGGTGGTTACCG913-924 CAACAAGGGTGAAGCAATCGGTGTTATCGCGGCACAGTCCA TCGGTGAACCGGGTA1319-1327 AACCGAGTCCTTCATCTCCGCGGCATCGTTCCAGGAGACCA CTCGC 1347-1360CTGCGCGGCCTGAAAGAGAACGTTATCGTGGGTCGTCTGAT CCCGGCAGGTACCGGTTACGC rpoB1248-1256 GCACGCGCGTTCCACCGGTTCTTACAGCCTGGTTACTCAGC AGCCGCTGG 1257-1262GGTTACTCAGCAGCCGCTGGGTGGTAAGGCACAGTTCG 1265-1274TAAGGCACAGTTCGGTGGTCAGCGTTTCGGGGAGATGGAAG TGTGGGCGC 1277-1287GGAAGTGTGGGCGCTGGAAGCATACGGCGCAGCATACACCC TGCAGGAAATGC 1288-1297ATACACCCTGCAGGAAATGCTCACCGTTAAGTCTGATGACG TGAACGGTC 1298-1310GTCTGATGACGTGAACGGTCGTACCAAGATGTATAAAAACA TCGTGGACGGCAACCATC 1311-1321CATCGTGGACGGCAACCATCAGATGGAGCCGGGCATGCCAG AATCCTTCAACG 1322-1329CATGCCAGAATCCTTCAACGTATTGTTGAAAGAGATTCGTT CGC

TABLE 3 Myx^(r) isolates from saturation mutagenesis and selectionnumber of amino acid independent MIC substitution isolates ratio rpoCsingle-substitution mutants  345 Lys→Arg 6 >32  345 Lys→Asn 24 32  345Lys→Thr 5 32 1351 Val→Phe 20 >32 Multiple-substitution mutants  345Lys→Asn; 451 Pro→Leu 1 1351 Val→Phe; 1356 Leu→Pro 5 1351 Val→Phe; 1357Ile→Met 5 1351 Val→Phe; 1359 Ala→Thr 2  345 Lys→Asn; 452 Leu→Pro; 1  453Val→Ala  318 Gly→Ala; 345 Lys→Asn; 1  430 His→Pro; 467 Ala→Gly rpoBsingle-substitution mutants 1255 Thr→Ile 1 2 1275 Val→Met 15 >32 1275Val→Phe 2 >32 1278 Leu→Val 2 2 1279 Glu→Lys 20 >32 1285 Tyr→Asp 1 2 1298Val→Leu 1 4 1315 Met→Leu 1 2 1317 Pro→Leu 2 2 1320 Pro→Ala 1 2 1322Ser→Thr 1 2 1322 Ser→Tyr 1 2 1322 Ser→Val 2 16 1325 Val→Leu 1 2Multiple-substitution mutants 1232 Met→Ile; 1275 Val→Met 1 1275 Val→Met;1298 Val→Leu 1 1278 Leu→Val; 1279 Glu→Lys 1 1279 Glu→Lys; 1285 Tyr→Asp 1c. Myx Requires a Binding Determinant in the Switch-Region Target.

Applicant has performed equilibrium binding experiments with wild-typeEscerichia coli RNAP and with [Arg345]β′-RNAP, an RNAP derivative havinga single-residue substitution within the bacterial RNAP homologousswitch-region amino-acid sequence (detecting binding of Myx to RNAP bymonitoring quenching by Myx of fluorescence emission of RNAP Trpresidues; methods analogous to those described in Yarbrough, et al.(1976) J. Biol. Chem. 15, 2669-2676). The results in FIG. 7 show thatsubstitution within the bacterial RNAP homologous switch-regionamino-acid sequence reduces binding of Myx, indicating that thebacterial RNAP homologous switch-region amino-acid sequence constitutesa binding determinant for Myx (as opposed to a conformationaldeterminant required for function of Myx but not for binding of Myx).

2. Myx: Mechanism of Transcription Inhibition

a. Myx Prevents Stable Interaction with Promoter DNA.

Applicant has performed transcription and DNA-binding experiments inorder to define the basic mechanism of transcription inhibition by Myx(methods as in Mukhopadhyay, et al. (2004) Mol. Cell 14, 739-751). Theresults indicate that Myx inhibits transcription by preventing stableinteraction of RNAP with promoter DNA—preventing either DNA binding, DNAretention, or both (sample data in FIG. 8).

b. Myx Prevents Stable Interaction with the Promoter-DNA Segment thatBinds within the RNAP Active-Center Cleft.

Applicant has performed DNA binding experiments with a series ofsubfragments of promoter DNA in order to map the interaction of RNAPwith promoter DNA inhibited by Myx (methods as in Mukhopadhyay, et al.(2004) Mol. Cell 14, 739-751). The results indicate that Myx inhibitsinteraction of RNAP with the segment of promoter DNA comprisingpositions −11 to +15 relative to the transcription start site (sampledata in FIG. 9). This DNA segment corresponds, precisely, to the DNAsegment proposed to bind within the RNAP active-center cleft, and to beaffected by clamp opening and closing, in structural models oftranscription initiation complexes (see Ebright (2000) J. Mol. Biol.304, 687-698; Murakami, et al. (2003) Curr. Opin. Stuct. Biol. 12,89-97; Borukhov & Nudler (2003) Curr. Opin. Microbiol. 6, 93-100;Murakami, et al. (2002) Science 296, 1285-1290; Naryshkin, et al. (2000)Cell 101, 601-611; Mekler, et al. (2002) Cell 108, 599-614).

3. Myx: Crystal Structure of RNAP-Myx Complex

Applicant has determined a crystal structure of T. thermophilus RNAPholoenzyme in complex with Myx. (Myx inhibits T. thermophilus RNAPholoenzyme with K_(i)=20 μM; unpublished data). Crystals of RNAP-Myxwere obtained by soaking pre-existing crystals of T. thermophilus RNAPholoenzyme in solutions containing Myx, x-ray diffraction data werecollected at the Brookhaven National Light Source beamline X-25, and thestructure was solved by molecular replacement (methods as in Tuske, etal. (2005) Cell 122, 541-552). The crystal structure has a resolution of3.0 Å (97.1% complete), an R factor of=0.253, and a free R factor of0.289.

The crystal structure defines the binding site for Myx (FIG. 10A),defines interactions between Myx and the binding site (FIG. 10B), andprovides a starting point for structure-based screening andstructure-based design for identification of new switch-region-targetinhibitors. The crystal structure establishes that Myx binds within thebacterial RNAP switch region, and, in particular, within the bacterialRNAP homologous switch-region amino-acid sequence (FIG. 10). Myx makesdirect interactions with the structural elements known as “switch 2” and“switch 1” (FIG. 10B; β′ residues 330-347 and 1319-1328, numbered as inEscerichia coli RNAP), and also makes direct interactions with adjacentsegments of the β′ and β subunits (FIG. 10B; β′ residues 1346-1357 and βresidues 1270-1292 and 1318-1328, numbered as in Escerichia coli RNAP).The interactions encompass all four segments of the bacterial RNAPhomologous switch-region amino acid sequence (β′ residues 345 and 1351,and β residues 1275-1292 and 1322-1326, numbered as in Escerichia coliRNAP; FIG. 10B). The interactions with switch 2 and switch 1 involveresidues conserved both in bacterial RNAP and in eukaryotic RNAP I, RNAPII, and RNAP III; the interactions with adjacent segments of β′ and βinvolve residues conserved in bacterial RNAP but not conserved ineukaryotic RNAP I, RNAP II, or RNAP III, consistent with the selectivityof Myx. Myx directly contacts all residues at which substitutionsconferring high-level Myx resistance are obtained (FIG. 10B).

Myx does not overlap the RNAP active center cleft or the predictedpositions of nucleic acids in transcription initiation and elongationcomplexes (FIG. 10A; Murakami, et al. (2002) Science 296, 1285-1290;Gnatt, et al. (2001) Science 292, 1876-1882; Westover, et al. (2004a)Science 303, 1014-1016; Westover, et al. (2004b) Cell 119, 481-489;Kettenberger, et al. (2004) Mol. Cell 16, 955-965; Naryshkin, et al.(2000) Cell 101, 601-611; Mekler, et al. (2002) Cell 108, 599-614).Indeed, Myx is nearly completely buried, with little or no surfaceaccessibility on the interior of the RNAP active-center cleft and withno surface accessibility on the on the exterior of RNAP (FIG. 10A).These observations suggest that Myx inhibits transcription throughallosteric interactions, not through direct, steric interactions.

The RNAP clamp in the crystal structure of RNAP-Myx adopts the sameconformation as in the crystal structure of unliganded RNAP in the samecrystal form (FIG. 10A; compare FIG. 1A): i.e., an intermediate clampconformation. This observation permits the conclusion that binding ofMyx is compatible with an intermediate clamp conformation. However, thisobservation does not permit the conclusion that binding of Myx favors,stabilizes, or induces an intermediate clamp conformation, since clampconformation in the crystal is constrained by, and may be determined by,crystal-lattice interactions.

The RNAP switch region in the crystal structure of RNAP-Myx adopts adifferent conformation from that in the crystal structure of unligandedRNAP in the same crystal form. The difference in conformation involves aseven-residue segment of switch 2—a seven-residue segment that differsin conformation in open-clamp, intermediate-clamp, and closed-clampconformational states (FIG. 1B; β′ residues 337-343, numbered as inEscerichia coli RNAP). The difference in conformation involves 1-4 Ådisplacements of Cα atoms of the seven-residue segment toward positionscharacteristic of those in the closed-clamp conformational state.

4. Myx: Working Hypothesis

While not wishing to be bound by any one hypothesis, Applicant infersfrom the genetic, biochemical, and structural results described above,that Myx may inhibit transcription by locking the RNAP switch region inone conformational state, preventing switch-region conformationalcycling, and thereby rendering the RNAP clamp unable to open to permitentry of DNA into the RNAP active-center cleft, unable to close topermit retention of DNA within the RNAP-active-center cleft, or both.

Example 2 Switch-Region-Target Inhibitors: Cor

The present example is directed to the use of corallopyronin (Cor) as asmall-molecule inhibitor of RNAP that, based on Applicant's discovery,inhibits RNAP through interaction with the bacterial RNAP homologousswitch-region amino-acid sequence. Cor is a polyketide-derived α-pyroneantibiotic structurally related to Myx, differing only by possession ofa seven-carbon side-chain extension (Irschik, et al. (1985) J. Antibiot.38, 145-152; FIG. 4A,B). Cor is produced by the myxobacteriumCorallococcus coralloides strain Cc c127 (DSM 2550; Irschik, et al.(1985) Supra). The compound potently inhibits growth of Gram-positiveand Gram-negative bacterial species, including Bacillus subtilis, B.megaterium, S. aureus, M. luteus, C. mediolanum, and Escerichia coliDH21tolC (MICs≦10 μg/ml for all; MIC≦0.1 μg/ml for S. aureus; Irschik,et al. (1985) Supra; unpublished data). The compound is bacteriocidal,as assessed in experiments with Escerichia coli DH21tolC (unpublisheddata). The compound inhibits bacterial RNAP (K_(i)=4 μM) but does notinhibit eukaryotic RNAP II (Irschik, et al. (1985) J. Antibiot. 38,145-152). The compound exhibits no cross-resistance with rifamycins,CBR-703, or microcin J25 (O'Neill, et al. (2000) Antimicrob. AgentsChemother 44, 3163-3166; unpublished data).

Applicant has tested Myx-resistant mutants for cross-resistance to thestructurally related antibiotic Cor and has performed saturationmutagenesis and directly isolated and characterized more than 75independent Cor-resistant mutants (methods as in Example 1, employingthe “doped” oligodeoxyribonucleotide primers in Table 2). The results,presented in Tables 4 and 5, establish that Cor functions throughinteractions with the same target as Myx: i.e., a target encompassingthe bacterial RNAP homologous switch-region amino-acid sequence. Inaddition, by defining a residue that interacts with theseven-carbon-atom side-chain extension present in Cor but absent in Myx(β residue 1326, numbered as in Escerichia coli RNAP), the resultsdefine the binding orientation of the ligand relative to the target,providing independent support for the binding orientation observed inthe crystal structure of the RNAP-Myx complex.

Applicant also has assessed effects of Cor on transcription, promoterbinding, and promoter-subfragment binding (methods as in Example 1). Theresults (not shown) establish that Cor functions through the samemechanism as Myx. TABLE 4 Myx/Cor/Rip cross-resistance patterns MICratio (MIC, mutant/ amino acid selected MIC, wild-type) substitutionresistance(s) Myx Cor Rip rpoC single-substitution mutants  345 Lys→ArgMyx, Cor, Rip >32 >8 >16  345 Lys→Asn Myx, Cor 32 8 8  345 Lys→Thr Myx,Cor 32 8 >16 1346 Gly→Asp Cor 1 4 4 1351 Val→Phe Myx, Cor,Rip >32 >8 >16 1352 Ile→Asn Rip 2 4 >16 1352 Ile→Ser Rip 2 4 >16 1354Gly→Cys Cor 2 2 4 rpoB single-substitution mutants 1255 Thr→Ile Myx 2 44 1275 Val→Met Myx, Cor >32 >8 8 1275 Val→Phe Myx >32 >8 >16 1278Leu→Val Myx 2 4 >16 1279 Glu→Gly Rip 32 4 >16 1279 Glu→Lys Myx, Cor >328 4 1283 Ala→Val Rip 2 4 4 1285 Tyr→Asp Myx 2 1 4 1291 Leu→Phe Myx, Rip2 1 4 1298 Val→Leu Myx 2 2 4 1315 Met→Leu Myx 2 2 4 1317 Pro→Leu Myx 2 22 1320 Pro→Ala Myx 2 4 4 1322 Ser→Pro Rip 32 4 >16 1322 Ser→Thr Myx 2 44 1322 Ser→Tyr Myx 2 4 2 1322 Ser→Val Myx 16 8 >16 1325 Val→Leu Myx 2 44 1326 Leu→Trp Rip 1 >8 >16

TABLE 5 Cor^(r) isolates from saturation mutagenesis and selectionnumber of amino acid independent MIC substitution isolates ratio rpoC 345 Lys→Arg 2 >8  345 Lys→Asn 13 8  345 Lys→Gln 2  345 Lys→Thr 3 8 1346Gly→Asp 1 4 1351 Val→Phe 15 >8 1354 Gly→Cys 2 2 rpoB single-substitutionmutants 1275 Val→Met 26 >8 1279 Glu→Lys 11 8 Multiple-substitutionmutants 1232 Met→Ile; 1275 Val→Met 1 1279 Glu→Lys; 1285 Tyr→Gly 1 1279Glu→Lys; 1287 Leu→Gln 1

Example 3 Switch-Region-Target Inhibitors: Rip

The present example is directed to the use of ripostatin (Rip) as asmall-molecule inhibitor of RNAP that, based on Applicant's discovery,inhibits RNAP through interactions with the bacterial RNAP homologousswitch-region amino-acid sequence. Rip is a polyketide-derivedmacrocylic-lactone antibiotic (Irschik, et al. (1995) J. Anitibiot. 48,787-792; Augustiniak, et al. (1996) Liebigs Ann. 10, 1657-1663); FIG.4C). Rip is produced by the myxobacterium Sorangium cellulosum strain Soce377 (DSM 7291; Irschik, et al. (1995) Supra). The compound potentlyinhibits growth of Gram-positive and Gram-negative bacterial species,including S. aureus, and Escerichia coli DH21tolC (MICs≦1 μg/ml;Irschik, et al. (1995) Supra; unpublished data). The compound isbacteriocidal, as assessed in experiments with Escerichia coli DH21tolC(unpublished data). The compound inhibits bacterial RNAP (K_(i)=0.3 μM)but does not inhibit eukaryotic RNAP II (Irschik, et al. (1995) Supra).The compound exhibits no cross-resistance with rifamycins, CBR-703, ormicrocin J25 (O'Neill, et al. (2000) Antimicrob. Agents Chemother. 44,3163-3166; Irschik, et al. (1995) Supra; unpublished data).

Applicant has tested Myx-resistant mutants for cross-resistance to thestructurally unrelated antibiotic Rip and has performed saturationmutagenesis and directly isolated and characterized more than 50independent Rip-resistant mutants (methods as in Example 1, employingthe “doped” oligodeoxribonucleotide primers in Table 2). The results,presented in Tables 4 and 6, establish that, surprisingly, in view ofits very different chemical structure (FIG. 4C; compare FIG. 4A), Ripfunctions through interactions with the same target as Myx: i.e., atarget encompassing the bacterial RNAP homologous switch-regionamino-acid sequence. Applicant also has assessed effects of Rip ontranscription, promoter binding, and promoter-subfragment binding(methods as in Example 1). The results (not shown) establish that,surprisingly, in view of its very different chemical structure (FIG. 4C;compare FIG. 4A), Rip functions through the same mechanism as Myx. TABLE6 Rip^(r) isolates from saturation mutagenesis and selection number ofamino acid independent MIC substitution isolates ratio rpoCsingle-substitution mutants  345 Lys→Arg 7 >16 1351 Val→Phe 9 >16 1352Ile→Asn 9 >16 1352 Ile→Ser 5 >16 multiple-substitution mutants 1349Glu→Asp; 1351 Val→Phe 1 1349 Glu→Asp; 1352 Ile→Ser 3 1350 Asn→Tyr; 1351Val→Phe 2 1351 Val→Phe; 1354 Gly→Cys 1 1351 Val→Phe; 1356 Leu→Pro 1 rpoBsingle-substitution mutants 1279 Glu→Gly 1 >16 1283 Ala→Val 1 4 1291Leu→Phe 1 4 1322 Ser→Pro 2 >16 1326 Leu→Trp 6 >16 multiple-substitutionmutants 1279 Glu→Val; 1283 Ala→Val 2 1322 Ser→Pro; 1325 Val→Gly 1 1323Phe→Cys; 1326 Leu→Trp 2 1328 Lys→Thr 1291 Leu→Phe; 1319 Met→Ile 1 1320Pro→Ser; 1321 Glu→Lys

Example 4 High-Throughput Assay for Switch-Region-Target Inhibitors

Applicant has developed and demonstrated a microplate-basedhigh-throughput assay for switch-region-target inhibitors. The assayemploys measurement of fluorescence-detected abortive initiation(methods analogous to those described in Mukhopadhyay, et al, (2004)Mol. Cell 14, 739-751; Tuske, et al. (2005) Cell 122, 541-552; see alsoSchlageck, et al., (1979) J. Biol. Chem. 254, 12074-12077). The assayinvolves two measurements performed in parallel: (i) measurement ofeffects on transcription by wild-type Escerichia coli RNAP, and (ii)measurement of effects on transcription by [Arg345]β′-RNAP, an RNAPderivative having a substitution within the switch-region target thatconfers high-level resistance to Myx, Cor, and Rip. Switch-region-targetinhibitors are identifiable as compounds that inhibit transcription incase i but do not inhibit transcription in case ii.

INDUSTRIAL APPLICABILITY

Compounds identified according to the target and method of thisinvention would have applications not only in antibacterial therapy, butalso in: (a) identification of bacterial RNAP (diagnostics,environmental-monitoring, and sensors applications), (b) labeling ofbacterial RNAP (diagnostics, environmental-monitoring, imaging, andsensors applications), (c) immobilization of bacterial RNAP(diagnostics, environmental-monitoring, and sensors applications), (d)purification of bacterial RNA polymerase (biotechnology applications),(e) regulation of bacterial gene expression (biotechnologyapplications), and (f) antisepsis (antiseptics, disinfectants, andadvanced-materials applications).

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

All patent and non-patent publications cited in this specification areindicative of the level of skill of those skilled in the art to whichthis invention pertains. All these publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated herein by reference.

1. A method for identifying an agent that binds to a bacterial RNAPhomologous switch-region amino-acid sequence in a first entity,comprising the steps of: (a) preparing a reaction solution including theagent to be tested and a first entity including a bacterial RNAPhomologous switch-region amino-acid sequence; and (b) detecting at leastone of the presence, extent, concentration-dependence, or kinetics ofbinding of the agent to the homologous switch-region amino-acidsequence.
 2. The method of claim 1 wherein the first entity is an intactbacterial RNAP.
 3. The method of claim 1 wherein the first entity is afragment of a bacterial RNAP.
 4. The method of claim 1 wherein the firstentity is Escerichia coli RNAP or a derivative thereof.
 5. The method ofclaim 1 wherein the first entity is Bacillus subtilis RNAP or aderivative thereof.
 6. The method of claim 1 further comprising the stepof: assessing at least one of the presence, extent,concentration-dependence, or kinetics of binding of the agent to asecond entity that contains a derivative of a bacterial RNAP homologousswitch-region amino-acid sequence having at least one substitution,insertion, or deletion.
 7. The method of claim 6 wherein the secondentity is a derivative of an intact bacterial RNAP.
 8. The method ofclaim 6 wherein the second entity is a derivative of a fragment of abacterial RNAP.
 9. The method of claim 6 wherein the second entity is aderivative of Escerichia coli RNAP.
 10. The method of claim 6 whereinthe second entity is a derivative of Bacillus subtilis RNAP.
 11. Themethod of claim 1 further comprising comparison of: (a) at least one ofthe presence, extent, concentration-dependence, or kinetics of bindingof the agent to the first entity, and (b) at least one of the presence,extent, concentration-dependence, or kinetics of binding of the agent toa eukaryotic RNAP derivative.
 12. The method of claim 11 wherein theeukaryotic RNAP derivative is a human RNAP derivative.
 13. The method ofclaim 11 wherein the eukaryotic RNAP derivative is a human RNAP IIderivative.
 14. A method for identifying an agent that inhibits anactivity of a bacterial RNAP by binding to a bacterial RNAP homologousswitch-region amino-acid sequence, comprising: (a) preparing a reactionsolution comprising the agent to be tested and a first entity containinga bacterial RNAP homologous switch-region amino-acid sequence; and (b)detecting at least one of the presence, extent,concentration-dependence, or kinetics of inhibition of an activity ofsaid first entity, wherein inhibition involves binding of the agent tothe bacterial RNAP homologous switch-region amino-acid sequence.
 15. Themethod of claim 14 wherein the first entity is an intact bacterial RNAP.16. The method of claim 14 wherein the first entity is a fragment of abacterial RNAP.
 17. The method of claim 14 wherein first entity isEscerichia coli RNAP or a derivative thereof.
 18. The method of claim 14wherein the first entity is Bacillus subtilis RNAP or a derivativethereof.
 19. The method of claim 14 wherein the activity istranscription initiation.
 20. The method of claim 14 wherein theactivity is transcription elongation.
 21. The method of claim 14 whereinthe activity is σ binding.
 22. The method of claim 14 wherein theactivity is DNA binding.
 23. The method of claim 14 wherein the activityis open-complex formation.
 24. The method of claim 14 wherein theactivity is RNA synthesis.
 25. The method of claim 14 further comprisingthe step of: assessing at least one of the presence, extent,concentration-dependence, or kinetics of the inhibition by the agent ofthe activity of a second entity that contains a derivative of abacterial RNAP homologous switch-region amino-acid sequence having atleast one substitution, insertion, or deletion.
 26. The method of claim25 wherein the second entity is a derivative of an intact bacterialRNAP.
 27. The method of claim 25 wherein the second entity is aderivative of a fragment of a bacterial RNAP.
 28. The method of claim 25wherein the second entity is a derivative of Escerichia coli RNAP. 29.The method of claim 25 wherein the second entity is a derivative ofBacillus subtilis RNAP.
 30. The method of claim 25 wherein the activityis transcription initiation.
 31. The method of claim 25 wherein theactivity is transcription elongation.
 32. The method of claim 25 whereinthe activity is open-complex formation.
 33. The method of claim 25wherein the activity is DNA binding.
 34. The method of claim 25 whereinthe activity is open-complex formation.
 35. The method of claim 25wherein the activity is RNA synthesis.
 36. The method of claim 25wherein inhibition of an activity of the first entity and inhibition ofan activity of the second entity are assessed sequentially.
 37. Themethod of claim 25 wherein inhibition of an activity of the first entityand inhibition of an activity of the second entity are assessedsimultaneously.
 38. The method of claim 14 further comprising comparisonof: (a) at least one of the presence, extent, concentration-dependence,or kinetics of inhibition by the agent of an activity of the firstentity, and (b) at least one of the presence, extent,concentration-dependence, or kinetics of inhibition by the agent of anactivity of a eukaryotic RNAP derivative.
 39. The method of claim 38wherein the eukaryotic RNAP derivative is a human RNAP derivative. 40.The method of claim 3 8 wherein the eukaryotic RNAP derivative is ahuman RNAP II derivative.
 41. The method of claim 14 wherein at leastone of the presence, extent, concentration-dependence, or kinetics ofinhibition by the agent of an activity of the first entity also iscompared to at least one of the presence, extent,concentration-dependence, or kinetics of inhibition by an inhibitorycompound specific to the bacterial RNAP homologous switch-regionamino-acid sequence of an activity of the first entity.
 42. A method foridentifying an agent that exhibits antibacterial activity by binding toa bacterial RNAP homologous switch-region amino-acid sequence,comprising: (a) preparing a reaction solution comprising the agent to betested and a first bacterium containing a bacterial RNAP homologousswitch-region amino-acid sequence; and (b) detecting inhibition of atleast one of viability of the bacterium and growth of the bacterium,wherein inhibition involves binding of the agent to the bacterial RNAPhomologous switch-region amino-acid sequence.
 43. The method of claim 42wherein the first bacterium is Escerichia coli or a derivative thereof.44. The method of claim 43 wherein the first bacterium is a tolC strainof Escerichia coli or a derivative thereof.
 45. The method of claim 44wherein the first bacterium is a tolC rfa strain of Escerichia coli or aderivative thereof.
 46. The method of claim 42 wherein the firstbacterium is Bacillus subtilis or a derivative thereof.
 47. The methodof claim 42 further comprising the step of: assessing inhibition by theagent of at least one of viability of a second bacterium and growth of asecond bacterium, said second bacterium containing a derivative of abacterial RNAP homologous switch-region amino-acid sequence having atleast one substitution, insertion, or deletion.
 48. The method of claim47 wherein the second bacterium is a derivative of Escerichia coli. 49.The method of claim 48 wherein the second bacterium is a derivative of atolC strain of Escerichia coli.
 50. The method of claim 49 wherein thesecond bacterium is a derivative of a tolC rfa strain of Escerichiacoli.
 51. The method of claim 47 wherein the second bacterium is aderivative of Bacillus subtilis.
 52. The method of claim 47 whereinantibacterial activity against the first bacterium and antibacterialactivity against the second bacterium are assessed sequentially.
 53. Themethod of claim 47 wherein antibacterial activity against the firstbacterium and antibacterial activity against the second bacterium areassessed simultaneously.
 54. The method of claim 42 whereinantibacterial activity of the agent against the first bacterium also iscompared to antibacterial activity of an inhibitory compound specific tothe bacterial RNAP homologous switch-region amino-acid sequence againstthe first bacterium.
 55. A method for identifying an agent that binds toa bacterial RNAP homologous switch-region amino-acid sequence,comprising (a) preparing a reaction solution comprising the agent to betested, a reference compound that binds to a homologous bacterial RNAPswitch-region amino-acid sequence, and a first entity containing abacterial RNAP homologous switch-region amino-acid sequence, and (b)detecting at least one of the presence, extent,concentration-dependence, or kinetics of competition by the agent forbinding of the reference compound to the homologous switch-regionamino-acid sequence.
 56. The method of claim 55 wherein the first entityis an intact bacterial RNAP.
 57. The method of claim 55 wherein thefirst entity is a fragment of a bacterial RNAP.
 58. The method of claim55 wherein the first entity is Escerichia coli RNAP or a derivativethereof.
 59. The method of claim 55 wherein the first entity is Bacillussubtilis RNAP or a derivative thereof.
 60. The method of claim 55wherein the reference compound contains a detectable group.
 61. Themethod of claim 55 wherein the detectable group contains a chromophore.62. The method of claim 55 wherein the detectable group contains afluorophore.
 63. The method of claim 55 wherein the reference compoundis a chromophore-labeledinhibitory compound specific to the bacterialRNAP homologous switch-region amino-acid sequence.
 64. The method ofclaim 55 wherein the reference compound is a fluorophore-labeledinhibitory compound specific to the bacterial RNAP homologousswitch-region amino-acid sequence.
 65. The method of claim 55 furthercomprising measurement of FRET.
 66. The method of claim 55 furthercomprising the step of: assessing at least one of the presence, extent,concentration-dependence, or kinetics of the binding of the agent to asecond entity that contains a derivative of a bacterial RNAP homologousswitch-region amino-acid sequence having at least one substitution,insertion, or deletion.
 67. The method of claim 66 wherein the secondentity is a derivative of an intact bacterial RNAP.
 68. The method ofclaim 66 wherein the second entity is a derivative of a fragment of abacterial RNAP.
 69. The method of claim 66 wherein the second entity isa derivative of Escerichia coli RNAP.
 70. The method of claim 66 whereinthe second entity is a derivative of Bacillus subtilis RNAP.
 71. Themethod of claim 55 further comprising comparison of: (a) at least one ofthe presence, extent, concentration-dependence, or kinetics of bindingof the agent to the first entity, and (b) at least one of the presence,extent, concentration-dependence, or kinetics of binding of the agent toa eukaryotic RNAP derivative.
 72. The method of claim 71 wherein theeukaryotic RNAP derivative is a human RNAP derivative.
 73. The method ofclaim 71 wherein the eukaryotic RNAP derivative is a human RNAP IIderivative.
 74. The method of claim 55 wherein at least one of thepresence, extent, concentration-dependence, or kinetics of binding ofthe agent to the first entity is compared to at least one of thepresence, extent, concentration-dependence, or kinetics of binding of aninhibitory compound specific to the bacterial RNAP homologousswitch-region amino-acid sequence to the first entity.
 75. A method foridentifying an agent that binds to a bacterial RNAP homologousswitch-region amino-acid sequence, comprising at least one of computerdocking and energy calculations with a first entity containing at leastone residue of a bacterial RNAP homologous switch-region amino-acidsequence.
 76. The method of claim 75 wherein the first entity is anintact bacterial RNAP.
 77. The method of claim 75 wherein the firstentity is a fragment of a bacterial RNAP.
 78. The method of claim 75wherein the first entity is an intact bacterial RNAP in complex with acompound specific for the switch-region target.
 79. The method of claim75 wherein the first entity is a fragment of a bacterial RNAP in complexwith a compound specific for the switch-region target.
 80. The method ofclaim 75 wherein first entity is Escerichia coli RNAP or a derivativethereof.
 81. The method of claim 75 wherein the first entity is Bacillussubtilis RNAP or a derivative thereof.
 82. The method of claim 75wherein the first entity is Thermus sp. RNAP or a derivative thereof.83. The method of claim 75 further comprising the step of: performing atleast one of computer docking and energy calculations with a secondentity containing at least one residue of a derivative of a bacterialRNAP homologous switch-region amino-acid sequence having at least onesubstitution, insertion, or deletion.
 84. The method of claim 83 whereinthe second entity is a derivative of an intact bacterial RNAP.
 85. Themethod of claim 83 wherein the second entity is a derivative of fragmentof a bacterial RNAP.
 86. The method of claim 83 wherein the secondentity is a derivative of an intact bacterial RNAP in complex with acompound specific for the switch-region target.
 87. The method of claim83 wherein the second entity is a derivative of a fragment of abacterial RNAP in complex with a compound specific for the switch-regiontarget.
 88. The method of claim 83 wherein second entity is a derivativeof Escerichia coli RNAP.
 89. The method of claim 83 wherein the secondentity is a derivative of Bacillus subtilis RNAP.
 90. The method ofclaim 83 wherein the second entity is a derivative of Thermus sp. RNAP.91. The method of claim 83 wherein computational analysis with the firstentity and computational analysis with the second entity are performedsequentially.
 92. The method of claim 83 wherein computational analysiswith the first entity and computational analysis with the second entityare performed simultaneously.
 93. The method of claim 75 furthercomprising comparison of: (a) results of at least one of computerdocking and energy calculations with the agent and a first entity, and(b) results of at least one of computer docking and energy calculationswith the agent and a eukaryotic RNAP derivative.
 94. The method of claim93 wherein the eukaryotic RNAP derivative is a human RNAP derivative.95. The method of claim 93 wherein the eukaryotic RNAP derivative is ahuman RNAP II derivative.
 96. The method of claim 93 wherein theeukaryotic RNAP derivative is a yeast RNAP derivative.
 97. The method ofclaim 93 wherein the eukaryotic RNAP derivative is a yeast RNAP IIderivative.
 98. The method of claim 75 wherein results of at least oneof computer docking and energy calculations with the agent and the firstentity also are compared to results of at least one of computer dockingand energy calculations with an inhibitory compound specific to thebacterial RNAP homologous switch-region amino-acid sequence and thefirst entity.